Abstract
Background
Quorum sensing (QS) is a cell density-based intercellular communication system that controls virulence gene expression and biofilm formation. In Pseudomonas aeruginosa (P. aeruginosa), the LasR system sits at the top of the QS hierarchy and coordinates the expression of a series of important traits. However, the role of lasR in phage infection remains unclear. This study aims to investigate the role of lasR QS in phage infection.
Methods
The P. aeruginosa phage was isolated from sewage, and its biological characteristics and whole genome were analyzed. The adsorption receptor was identified via a phage adsorption assay. Following lasR gene knockout, the adsorption rate and bactericidal activity of phage were analyzed. Finally, real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to explore how lasR promoting phage infection.
Results
The lytic phage vB_Pae_PLY was isolated and lipopolysaccharide (LPS) was identified as its adsorption receptor. The adsorption rate and bactericidal activity of vB_Pae_PLY were reduced after lasR knockout. RT-qPCR results showed that the expression of galU, a key gene involved in LPS synthesis, was down-regulated, and several genes related to type IV pili (T4P) were also down-regulated in the lasR mutant PaΔlasR.
Conclusions
The study showed that QS lasR may promote phage vB_Pae_PLY infection by involving in the synthesis of LPS and T4P. This study provides an example of QS in promoting phage infection and deepens the understanding of phage-bacteria interactions.
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Background
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that uses quorum sensing (QS) signaling molecules to regulate the expression of virulence genes and biofilm [1]. P. aeruginosa has a particularly complex QS signaling network, which primarily consists of four interconnected systems (LasI/R, RhlI/R, PQS, and IQS) [2]. In the Las and Rhl systems, the lasI and rhll genes are responsible for the synthesis of N -(3-oxo-dodecanoyl)- L -homoserine lactone (3- oxo -C12-HSL) and N -(butanoyl)- L -homoserine lactone (C4-HSL), respectively. Then, 3- oxo -C12-HSL and C4-HSL interact with LasR, respectively, to form LasR- 3- oxo -C12-HSL and RhlR- C4-HSL complex to regulate target genes [3]. The PQS system enables the biosynthesis of QS signal molecules 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1 H)-quinolone (PQS, Pseudomonas quinolone signal) [4]. IQS is the fourth and recently discovered QS system, which synthesizes the signaling molecule 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) [1]. Among all the QS systems, the Las tops the signal network. The LasR- 3- oxo -C12-HSL complex positively regulates the expression of receptor and synthase genes in the downstream QS system, thus establishing a regulatory feedforward loop [5]. QS directly or indirectly regulates more than 10% of its genome and about 20% of its bacterial proteome [6, 7], and controls the expression of a series of important traits in P. aeruginosa [8, 9].
Phages are viruses that specifically infect bacteria and are abundant in environments associated with their host bacteria. Phages bind to the surfaces of bacterial cells by recognizing adsorption receptors [50].
RT-qPCR analysis in the present study revealed that galU and T4P-related genes (pilA, pilB, pilC and pilD, pilQ and pilV, pilW and pilY) were significantly decreased in the PaΔlasR (Fig. 8). LPS is composed of O antigen, core oligosaccharide, and lipid A. galU encodes UDP-glucose pyrophosphorylase (GalU), and was found to be involved in the synthesis of the core region of P. aeruginosa LPS [43, 51]. The current study demonstrates that the phage vB_Pae_PLY utilizes surface LPS as the receptor for adsorption (Fig. 6). Thus, the reduced phage sensitivity in PaΔlasR can be largely attributed to the impaired LPS synthesis. In addition, pili also play an important role in phage infection; therefore, downregulation of T4P may also be another reason for reduced phage sensitivity. LPS and T4P are important virulence factors for the opportunistic pathogen P. aeruginosa. On the other hand, serving as phage receptors, LPS and T4P facilitate phage infection with their synthesis being positively regulated by QS. Therefore, lasR QS inhibition will affect the synthesis of bacterial LPS and T4P, resulting in reduced phage adsorption with LPS and T4P as receptors, and weaken the bactericidal effect of phages to a certain extent.
Conclusions
In conclusion, this study demonstrates that lasR may promote phage infection by positively regulating the biosynthesis of LPS and T4P. Disrupting lasR expression leads to decreased phage sensitivity. Given the complex and multifaceted role of QS in host-phage interactions, the future research is needed to uncover the various mechanisms by which QS participates in phage infection.
Data availability
The complete genome sequence of phage vB_Pae_PLY presented in this study is openly available in [GenBank, OR689712].
Abbreviations
- Pseudomonas aeruginosa :
-
P. aeruginosa
- QS:
-
Quorum sensing
- RT-qPCR:
-
Real-Time quantitative polymerase chain reaction
- LPS:
-
Lipopolysaccharide
- T4P:
-
Type IV pili-related
- AHL:
-
N-acylhomoserine lactones
- 3-oxo-C12-HSL:
-
N -(3-oxo-dodecanoyl)- L -homoserine lactone
- C4-HSL:
-
N -(butanoyl)- L -homoserine lactone
- HHQ:
-
2-heptyl-4-hydroxyquinoline
- PQS:
-
2-heptyl-3-hydroxy-4(1 H)-quinolone
- IQS:
-
2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde
- LB:
-
Luria-Bertani
- Car:
-
Carbenicillin
- Gen:
-
Gentamicin
- PFU:
-
Plaque-forming unit
- CFU:
-
Colony-forming unit
- VFDB:
-
Virulence Factor Database
- CARD:
-
Comprehensive Antibiotic Resistance Database
- PCR:
-
Polymerase chain reaction
- PBS:
-
Phosphate-buffered saline
- MOI:
-
Multiplicity of infection
- TEM:
-
Transmission electron microscopy
- CDS:
-
Coding DNA sequences
- ICTV:
-
International Committee on Taxonomy of Viruses
- OmpK:
-
Outer membrane protein K
- GalU:
-
UDP-glucose pyrophosphorylase
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Funding
This work was supported by the Health Department of Zhejiang Province of the People’s Republic of China, No. 2022KY895; the research grants from the National Natural Science Foundation of China, No. 82172328; and the Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, No. 2022E10022.
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YL and ZCY performed the experiments, generated figures, analyzed the data, and drafted the manuscript under the supervision of QW and TLZ. ZZM, MRT, HCC, CRQ, and WLZ conducted the data analysis. The research plan for this project was conceived based on several rounds of discussions among all co-authors. All authors read and approved the final version of the manuscript.
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All the experimental methods were performed in accordance with approved guidelines and regulations. The First Affiliated Hospital of Wenzhou Medical University’s ethics committee authorized each investigative protocol used in this study. Informed consent was obtained from all participants.
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Liu, Y., Yao, Z., Mao, Z. et al. Quorum sensing gene lasR promotes phage vB_Pae_PLY infection in Pseudomonas aeruginosa. BMC Microbiol 24, 207 (2024). https://doi.org/10.1186/s12866-024-03349-7
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DOI: https://doi.org/10.1186/s12866-024-03349-7