Transcriptome Analysis Reveals the Resistance Mechanism of Pseudomonas aeruginosa to Tachyplesin I

Abstract

Background: Tachyplesin I is a cationic antimicrobial peptide with a typical cyclic antiparallel β-sheet structure. We previously demonstrated that long-term continuous exposure to increased concentration of tachyplesin I can induce resistant Gram-negative bacteria. However, no significant information is available about the resistance mechanism of Pseudomonas aeruginosa (P. aeruginosa) to tachyplesin I.

Materials and methods: In this study, the global gene expression profiling of P. aeruginosa strain PA-99 and P. aeruginosa CGMCC1.2620 (PA1.2620) was conducted using transcriptome sequencing. For this purpose, outer membrane permeability and outer membrane proteins (OMPs) were further analyzed.

Results: Transcriptome sequencing detected 672 upregulated and 787 downregulated genes, covering Clusters of Orthologous Groups (COGs) of P. aeruginosa strain PA-99 compared with PA1.2620. Totally, 749 differentially expressed genes (DEGs) were assigned to 98 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and among them, a two-component regulatory system, a beta-lactam resistance system, etc. were involved in some known genes resistant to drugs. Additionally, we further attempted to indicate whether the resistance mechanism of P. aeruginosa to tachyplesin I was associated with the changes of outer membrane permeability and OMPs.

Conclusion: Our results indicated that P. aeruginosa resistant to tachyplesin I was mainly related to reduced entry of tachyplesin I into the bacterial cell due to overexpression of efflux pump, in addition to a decrease of outer membrane permeability. Our findings were also validated by pathway enrichment analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR). This study may provide a promising guidance for understanding the resistance mechanism of P. aeruginosa to tachyplesin I.

Keywords: P. aeruginosa; antibiotic resistance; differentially expressed genes; outer membrane permeability; outer membrane proteins; tachyplesin I.

Figure 1

Hierarchical clustering and Volcano plot of differential expression in PA1.2620 original strain vs PA-99 mutant. (A). Hierarchical clustering. T04-T05 stands for two biological duplications of PA1.2620 strain, T09-T11 stands for three biological duplications of tachyplesin I-resistant PA-99 strain. Each column represents one sample, and each row represents one gene. The red and green gradients indicate an increase and a decrease in gene expression abundance, respectively. (B). Volcano plot. Each point in the volcano plot represents one gene, and the abscissa represents the logarithm of the expression difference fold of one gene in PA1.2620 original strain vs PA-99 mutant. This vertical axis represents the negative logarithm of the error detection rate. The green dots represent downregulated genes, the red dots represent upregulated genes and the black dots represent unchanged genes.

Figure 2

Functional annotation of DEGs in PA1.2620 original strain vs tachyplesin-resistant PA-99 mutant. (A). GO enrichment categorization; (B). COG classification; (C). KEEG categorization.

Figure 3

Illustrating the RT-qPCR conformation results for the 10 selected DEGs. The X-axis represents the selected 10 genes and the Y-axis represents the fold change values derived from RNA-seq and RT-qPCR.

Figure 4

Outer membrane permeability of original and mutant strains.

Figure 5

Analysis of OMPs of different P. aeruginosa strains. (A). 1, 4: PA1.2620; 2–3: PA-99. (B). 1, 4: PA27853; 2–3: PA-88. Abbreviation: M, standard protein marker.