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. 2023 Mar 21;2(1):28-42.
doi: 10.1002/mlf2.12059. eCollection 2023 Mar.

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system

Xi Liu  1   2   3 , Minlu Jia  2 , Jing Wang  2 , Hang Cheng  2 , Zhao Cai  2 , Zhaoxiao Yu  1 , Yang Liu  4 , Luyan Z Ma  1 , Lianhui Zhang  3 , Yingdan Zhang  2   5 , Liang Yang  2   5
Affiliations

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system

Xi Liu et al. mLife. .

Abstract

Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs. In this study, a high-throughput transposon-insertion site sequencing (Tn-seq) approach was employed to identify novel P. aeruginosa biofilm genetic determinants. When analyzing the novel biofilm regulatory genes, we found that the cell division factor ZapE (PA4438) controls the P. aeruginosa pqs quorum sensing system. The ∆zapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) had been reduced. Further biochemical analysis showed that ZapE interacts with PqsH, which encodes the synthase that converts 2-heptyl-4-quinolone (HHQ) to PQS. In addition, site-directed mutagenesis of the ATPase active site of ZapE (K72A) abolished the positive regulation of ZapE on PQS signaling. As ZapE is highly conserved among the Pseudomonas group, our study suggests that it is a potential drug target for the control of Pseudomonas infections.

Keywords: Pseudomonas aeruginosa; Pseudomonas quinolone signal; ZapE; biofilm; transposon‐insertion site sequencing (Tn‐seq).

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Transposon‐insertion site sequencing analysis of genetic determinants for Pseudomonas aeruginosa biofilm formation. (A) Correlation analysis of input and output samples. (B) Quality control analysis of two inputs from the same batch of experiments. Each point represents the number of unique gene reads (UGRs) of one specific mutagenized gene in the duplicate samples. (C) Quality control analysis of two outputs from different batches of experiments. (D) Dot plot showing the significantly reduced colonization of mutants during biofilm formation. Each dot represents the normalized UGRs of each gene in input and output. Fold change (FC) is colorized to show the differences. (E) Functional categories of essential genes during biofilm formation and maintenance. Genes were deemed as essential factors during P. aeruginosa biofilm formation, in the scenario that all mutants impart no fitness cost, when log2|FC (input/output)| > 2 and p < 0.05.
Figure 2
Figure 2
Biofilm formation by the mutants of five candidate biofilm determinants (PA0222, PA1112, PA2345, PA3797, and PA4438) with unknown functions compared to wild‐type strain PAO1. (A) Confocal laser scanning microscope images showing biofilms formed by PAO1, ΔPA0222, ΔPA1112, ΔPA2345, ΔPA3797, and ΔPA4438. Biofilms were stained by SYTORED17. Scale bar = 50 μm. (B) Proportion of PAO1 (tagged with mCherry) and mutants (tagged with GFP) in cocultured biofilms. ***p < 0.001. (C) Growth curve of PAO1, ΔPA0222, ΔPA1112, ΔPA2345, ΔPA3797, and ΔPA4438.
Figure 3
Figure 3
ZapE is required for proper cell division under high‐temperature and oxygen‐limited conditions and biofilm formation. (A) Morphology of PAO1, ΔPA4438, its zapE complementation strain ΔPA4438/pzapE and Escherichia coli zapE complementation strain ΔPA4438/peczapE were cultivated in 37°C aerobic, 42°C aerobic, and 37°C anaerobic environment. The white arrows point to the elongated cells. Scale bar = 5 μm. (B) Biofilm morphology of PAO1, ΔzapE, ΔzapE/pzapE, and ΔzapE/peczapE under 37°C aerobic condition. Biofilms were stained by SYTORED17. Scale bar = 20 μm.
Figure 4
Figure 4
Expression of genes related to phenazine synthesis, pyoverdine synthesis, and quorum sensing is significantly downregulated in ΔzapE compared to PAO1. (A) Functional categories of significantly dysregulated genes (log2|FC (ΔzapE/PAO1)| > 2 and p< 0.05). (B) Dysregulated genes involved in quorum sensing, pyoverdine synthesis, and phenazine synthesis.
Figure 5
Figure 5
ZapE enhances the synthesis of PQS, pyocyanin, and pyoverdine. (A) Production of pyocyanin of PAO1/pUCP20, ΔzapE/pUCP20, ΔzapE/pzapE, and ΔzapE/pK72A. (B) Production of pyoverdine of PAO1/pUCP20, ΔzapE/pUCP20, ΔzapE/pzapE, and ΔzapE/pK72A. (C) Maximum pqsA expression of PAO1, ΔzapE, and ΔzapE::zapE at different concentrations of HHQ. HHQ was exogenously added into cultures. ***p < 0.001. HHQ, 2‐heptyl‐4‐quinolone; ns, no significant.
Figure 6
Figure 6
ZapE is required for proper cell division and biofilm formation of PA14 and PAK. (A) Morphology of PA14, PA14ΔzapE, PA14ΔzapE/pzapE, PAK, PAKΔzapE, and PAKΔzapE/pzapE in 37°C aerobic, 42°C aerobic, and 37°C anaerobic environment. The white arrows point to the elongated cells. Scale bar = 5 μm. (B) Biofilm morphology of PA14, PA14ΔzapE, PA14ΔzapE/pzapE, PAK, PAKΔzapE, and PAKΔzapE/pzapE under 37°C aerobic condition. The biofilm was stained by SYTO9. Scale bar = 50 μm.
Figure 7
Figure 7
Model of the ZapE regulation in the pqs quorum sensing system in P. aeruginosa. HHQ is synthesized by PqsABCD and converted to PQS by PqsH. ZapE is a cell division factor, which can directly interact with PqsH and positively regulate the synthesis of PQS. Both HHQ and PQS can bind to the transcriptional regulator PqsR; however, the affinity of PQS is approximately 100‐fold more potent than HHQ at stimulating PqsR activity. Autoinduction occurs when either HHQ or PQS binds to PqsR, and then the expression of pqsA‐E operon is activated. PqsE is a putative metallohydrolase protein, which positively regulates pyocyanin biosynthesis as well as biofilm formation. The biosynthesis of pyoverdine can be positively impacted by the biofilm and the iron chelator PQS. In brief, ZapE regulates P. aeruginosa biofilm formation by impacting the pqs quorum sensing.
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