Deletion of the PA4427-PA4431 Operon of Pseudomonas aeruginosa PAO1 Increased Antibiotics Resistance and Reduced Virulence and Pathogenicity by Affecting Quorum Sensing and Iron Uptake.

Lixin Shen,Shiwei Wang,Liping Wang,Yuqi Feng,Jian Zhang,Yulu Wang,Lang Gao,Mengjiao Yang

doi:10.3390/microorganisms9051065

Abstract

The respiratory chain is very important for bacterial survival and pathogenicity, yet the roles of the respiratory chain in P. aeruginosa remain to be fully elucidated. Here, we not only proved experimentally that the operon PA4427-PA4431 of Pseudomonas aeruginosa PAO1 encodes respiratory chain complex III (cytobc1), but also found that it played important roles in virulence and pathogenicity. PA4429–31 deletion reduced the production of the virulence factors, including pyocyanin, rhamnolipids, elastase, and extracellular polysaccharides, and it resulted in a remarkable decrease in pathogenicity, as demonstrated in the cabbage and Drosophila melanogaster infection models. Furthermore, RNA-seq analysis showed that PA4429–31 deletion affected the expression levels of the genes related to quorum-sensing systems and the transport of iron ions, and the iron content was also reduced in the mutant strain. Taken together, we comprehensively illustrated the function of the operon PA4427–31 and its application potential as a treatment target in P. aeruginosa infection.

Highlights

The respiratory chain, including the aerobic and anaerobic respiratory branches, is one of the most key supplying energy pathways for cellular metabolism
The genes PA4427, PA4428, PA4429, PA4430, and PA4431 were predicted to constitute an operon according to the P. aeruginosa genome database, and it was consistent with the RT-PCR results (Figure 1A)
The disk diffusion test showed that, compared with the wildtype strain PAO1, all the mutants exhibited smaller inhibition zones under the treatment condition of four kinds of the tested aminoglycoside antibiotics, including kanamycin, gentamycin, tobramycin, and amikacin (Figure 1B). Their minimum inhibitory concentrations (MICs) values were determined, and the results showed that the MIC values of the single-gene deletion or the three-gene deletion were all increased threefold for the four antibiotics, indicating a resistance increase for the aminoglycoside antibiotics of these mutants (Table 3)

Summary

Introduction

The respiratory chain, including the aerobic and anaerobic respiratory branches, is one of the most key supplying energy pathways for cellular metabolism. The shared main respiratory equipment of the aerobic and anaerobic respiration pathways contains NADH dehydrogenase (complexes I), coenzyme Q, cytochrome bc complex (complex III), and cytochrome c. In Pseudomonas aeruginosa, the aerobic chain contains terminal oxidases (complex IV, e.g., panthenol and cytochrome c oxidases), and the anaerobic respiratory chain possesses a series of NOx reductases (e.g., nitrate, nitrite, nitric oxide, and nitrous oxide reductases) [1]. Protons are pumped out across the bacterial membrane during electron transfer through complexes I, III, and IV, which form the proton gradient for ATP synthesis [2]. The bc complex in the center of the respiratory chain is responsible for the electrons transferring from coenzyme Q to cytochrome c [3]. All bc complexes contain three redox subunits: cyt b (two b-type hemes: bL and bH ), cyt c1 (a c-type heme), and ISP (two Fe-S clusters) [4]

Methods

Results

Conclusion