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Additive Effects of Quorum Sensing Anti-Activators on Pseudomonas aeruginosa Virulence Traits and Transcriptome
Kyle L Asfahl1, Martin Schuster1
1Department of Microbiology, Oregon State University, Corvallis, OR, United States.
Abstract:
In the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing (QS) via acyl-homoserine lactone (AHL) signals coordinates virulence gene expression. AHL signals must reach a critical threshold before enough is bound by cognate regulators LasR and RhlR to drive transcription of target genes. In addition, three anti-activator proteins, QteE, QscR, and QslA, sequester QS regulators to increase the threshold for induction and delay expression of QS target genes. It remains unclear how multiple anti-activators work together to achieve the quorum threshold. Here, we employed a combination of mutational, kinetic, phenotypic, and transcriptomic analysis to examine regulatory effects and interactions of the three distinct anti-activators. We observed combinatorial, additive effects on QS gene expression. As measured by reporter gene fusion, individual deletion of each anti-activator gene increased lasB expression and QS-controlled virulence factor production. Deletion of qslA in combination with the deletion of any other anti-activator gene resulted in the greatest increase and earliest activation of lasB gene expression. Western analysis revealed that relative increases in soluble LasR in anti-activator mutants correlate with increased lasB expression and QS-controlled virulence factor production. RNA-seq of the previously uncharacterized QslA and QteE regulons revealed overlapping, yet distinct groups of differentially expressed genes. Simultaneous inactivation of qteE and qslA had the largest effect on gene expression with 999 genes induced and 798 genes repressed in the double mutant vs. wild-type. We found that LasR and RhlR-activated QS genes formed a subset of the genes induced in the qteE, qslA, and double mutant. The activation of almost all of these QS genes was advanced from stationary phase to log phase in the qteE qslA double mutant. Taken together, our results identify additive effects of anti-activation on QS gene expression, likely via LasR and RhlR, but do not rule out QS-independent effects.
Insights
Pseudomonas aeruginosa quorum sensing (QS) uses anti-activator proteins to delay virulence gene expression. Removing these proteins, especially QslA and QteE, accelerates QS gene activation and virulence factor production.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Gene Regulation
Background:
- Quorum sensing (QS) in *Pseudomonas aeruginosa* coordinates virulence through acyl-homoserine lactone (AHL) signals, requiring a threshold concentration for gene activation.
- Anti-activator proteins (QteE, QscR, QslA) increase the QS induction threshold by sequestering QS regulators, delaying virulence gene expression.
- The combined regulatory mechanisms of these multiple anti-activators remain incompletely understood.
Purpose of the Study:
- To investigate the regulatory effects and interactions of the three anti-activator proteins (QteE, QscR, QslA) in *Pseudomonas aeruginosa*.
- To elucidate how these anti-activators collectively influence quorum sensing (QS) and virulence gene expression.
- To characterize the regulons of QslA and QteE and their impact on QS-dependent and independent gene expression.
Main Methods:
- Utilized mutational analysis, including single and double anti-activator gene deletions.
- Employed reporter gene fusions to quantify *lasB* expression.
- Performed Western analysis to assess LasR protein levels.
- Conducted RNA-sequencing (RNA-seq) to analyze global gene expression changes in anti-activator mutants.
Main Results:
- Individual deletion of anti-activator genes increased *lasB* expression and virulence factor production, indicating additive effects.
- Simultaneous deletion of *qslA* with other anti-activator genes resulted in the greatest and earliest activation of *lasB*.
- RNA-seq revealed that the double mutant *qteE qslA* significantly altered the expression of 999 induced and 798 repressed genes.
- QS-regulated genes were a subset of those induced in the *qteE qslA* double mutant, with activation advanced to the log phase.
Conclusions:
- Anti-activation by QteE, QscR, and QslA exhibits combinatorial and additive effects on QS gene expression, primarily mediated through LasR and RhlR.
- The inactivation of *qteE* and *qslA* significantly advances the timing of QS gene activation.
- While QS-dependent effects are evident, the study does not exclude potential QS-independent roles for these anti-activators.
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