Novel Insight of Transcription Factor PtrA on Pathogenicity and Carbapenems Resistance in Pseudomonas aeruginosa

Abstract

Introduction: Globally, Pseudomonas aeruginosa (PA) is emerging as a predominant nosocomial pathogen that often induces aggressive and even deadly infections. Pseudomonas type III repressor A (PtrA) can be activated specifically by copper ions and interacts with type-III transcriptional activator ExsA. This study aims to provide insight into the PtrA-mediated regulation of the pathogenicity and antibiotics resistance of PA.

Methods and results: The results of transcriptome sequencing analyses and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) showed that PtrA plays a dual regulatory role in the virulence systems of PA: negatively regulates the type-III secretion system (T3SS) and positively regulates the quorum-sensing system (QS). The ptrA mutant attenuated extracellular virulence related to QS like pyocyanin, elastase, rhamnolipids, proteolytic activity, and biofilm production. According to adhesion and invasion experiments, PtrA can not only contribute to the adhesiveness but also the invasive of PA. Moreover, the PtrA-mediated regulation of PA pathogenicity was determined both in vivo and in vitro through cytotoxicity and Galleria mellonella survival experiments. In addition, apart from virulence, PtrA was found to influence the carbapenems resistance of PA. After deleting ptrA, the minimum inhibitory concentration (MIC) of carbapenems antibiotics was decreased by 2-fold, while a 2-8 fold increase was noted for the complemented strain.

Conclusion: Our findings establish that PtrA exerts a regulatory role in both pathogenicity and carbapenems resistance of PA. This work may shed light on a novel target for the clinical treatment of PA.

Keywords: Pseudomonas aeruginosa; PtrA; Quorum-sensing; T3SS; carbapenems resistance; pathogenicity.

Figure 1

Transcriptome sequencing analysis of ΔptrA and PAO1 strain. (A) The volcano plot of DEGs. (B) The KEGG pathway classification enrichment of DEGs. (C) STRING analysis of 290 downregulated and 87 upregulated DEGs; Lines represent reported or estimated protein-protein associations, and the nodes of genes involved in conducting similar functions are circled in black.

Figure 2

The relative quantitative analyses of T3SS, and QS-related genes. (A) The effect of PtrA on T3SS gene. (B) The effect of PtrA on QS gene. The gene expression level in WT strain was adjusted to 1.0 (presented as a dotted line). ns, not statistically significant, *P < 0.05, **P < 0.01, and***P < 0.001 analyzed via Student’s t-test. The results are expressed as mean ± SD, and the data represent a minimum of 3 individual experiments.

Figure 3

Effect of PtrA mutation on motility. ns, not statistically significant, *P < 0.05, **P < 0.01, and***P < 0.001 analyzed via Student’s t-test. The results are expressed as mean ± SD, and the data represent a minimum of 3 individual experiments.

Figure 4

Effect of PtrA on QS-related extracellular virulence factor. (A and C) The determination of elastase production. (B and D) The determination of pyocyanin production. (E) The determination of rhamnolipid production. (F) The determination of proteolytic activity. ns, not statistically significant, *P < 0.05, **P < 0.01, and***P < 0.001 analyzed via Student’s t-test. The results are expressed as mean ± SD, and the data represent a minimum of 3 individual experiments.

Figure 5

PtrA affected planktonic growth and biofilm formation. (A) Growth curve assay. (B and C) Biofilm formation assay. **P < 0.01, and ***P < 0.001 analyzed via Student’s t-test. The results are expressed as mean ± SD, and the data represent a minimum of 3 individual experiments.

Figure 6

Analysis of the adherence and invasion ability of PA. (A) Adherence; (B) Invasion. ns, not statistically significant, *P < 0.05, and ***P < 0.001. The results are expressed as mean ± SD, and the data represent a minimum of 3 individual experiments.

Figure 7

PtrA is crucial for PA virulence. (A) Cytotoxicity assay. (B) Galleria mellonella experiments. ns, not statistically significant, *P < 0.05, and**P < 0.01 via Student’s t-test. The results are expressed as mean ± SD, and the data represent a minimum of 3 individual experiments.