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

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.

Keywords: antibiotic resistance; cytochrome bc1 (cytbc1); iron transport; quorum-sensing systems; respiratory chain; virulence and pathogenicity.

Figure 1

The transcription profiles of PA4427-PA4431 and the antibiotic susceptibility change after their mutation. (A) The products of PCR and RT-PCR using different primers and templates. The gray box between PA4428 and PA4429 is the intergenic region. Lane 1: r1 and r2 primers and genomic DNA template; Lane 2: r1 and r2 primers and cDNA template; Lane 5: r5 and r6 primers and genomic DNA template; Lane 6: r5 and r6 primers and cDNA template; Lane 9: r3 and r4 primers and genomic DNA template; Lane 10: r3 and r4 primers and cDNA template; Lanes 3, 7, and 11: non-retro-transcribed mRNA template as a negative control; and Lanes 4 and 8: 1 kb ladder marker. (B) Antibiotic susceptibility of PAO1, PAO1(Δ4429), PAO1(Δ4430), and PAO1(Δ4431) in LB plates with different antibiotics. Kan: kanamycin; Amk: amikacin; Gm: gentamicin; Tob: tobramycin. (C) The biofilm was examined in the PAO1, PAO1(ΔPA4428), PAO1(ΔPA4429), PAO1(ΔPA4430), PAO1(ΔPA4431), and PAO1(ΔPA4429–31) strains. The error bars represent standard errors. **** significantly different, p < 0.001.

Figure 2

The physiological effects after PA4427–PA4431 mutation. (A,B) The effect of nitrate and nitrite on the bacterial growth. (C) ATP detection in the various strains. (D,E) The effect of the gene pqsR and the molecule HQNQ on the bacterial growth in the LB broth. The error bars represent standard errors. * p < 0.05. **** p < 0.001.

Figure 3

The effects of PA4429–31 deficiency on pyocyanin production. (A,B) The transcription levels of phzA1 and phzA2 in the wild-type strain PAO1 and the mutants. (B) The content of pyocyanin in the wild-type strain PAO1 and the mutants. The error bars represent standard errors. * p < 0.05.

Figure 4

The effects of PA4429–31 deficiency on rhamnolipids (A) and polysaccharides (B) in the wild-type strain PAO1 and the mutants. (A) Rhamnolipid contents were normalized by the OD₆₀₀ of the bacteria. (B) The strains were cultured in the plates with Congo Red. The error bars represent standard errors. * p < 0.05.

Figure 5

The effects of PA4429–31 deficiency on virulence and motility. (A) Expression levels of elastase in the wild-type strain PAO1 and the mutants. (B) Swarming, swimming, and twitching of the wild-type strain PAO1 and the mutants were detected. The error bars represent standard errors. * p < 0.05.

Figure 6

The deficiency of PA4429–31 has influenced bacterial pathogenicity. (A) The decay area of PAO1 and PAO1(ΔPA4429–31) in the infection model of Chinese cabbage. (B) The mortality of PAO1 and PAO1(ΔPA4429–31) in the infection model of Drosophila melanogaster.

Figure 7

The transcriptional profiles of the genes related to the quorum sensing system, iron metabolism and the iron concentration in bacterial cells. (A–D) The transcriptional levels of lasI, lasR, pqsH, and rhlR were detected through the CPS (count per second) values. (E,F) The transcriptional levels of tonB1 and fur were detected through the CPS values and RNA-Seq analysis. The FPKM (fragments per kilobase of exon model per million mapped fragments) is the normalized number of transcripts measured for each gene. (G) The content of iron was tested through ICP-MS in PAO1 and PAO1(Δ4429–31). (H) The ROS was detected by using 2’, 7’-dichlorofluorescein diacetate (carboxy-H2DCFDA) in PAO1 and PAO1(Δ4429–31). RFU: relative fluorescence units. The error bars represent standard errors. ** p < 0.01. *** p < 0.001. **** p < 0.0001.