Additive Effects of Quorum Sensing Anti-Activators on Pseudomonas aeruginosa Virulence Traits and Transcriptome

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.

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

“…The activated LuxR-type receptors then alter gene expression levels to initiate behaviors that will benefit the group and/or are only achievable as a bacterial community, including a range of virulence phenotypes such as biofilm formation, protease and toxin production, and motility mechanisms (Lyczak et al, 2000). The pathogen P. aeruginosa has a fairly complex QS circuit involving three LuxR-type receptors (LasR, RhlR, and QscR) (Asfahl and Schuster, 2018), two AHL signals ( N -(3-oxo)-dodecanoyl HL, OdDHL [compound 1 in Fig. 1A] and N -butyl HL, BHL) produced by LuxI-type synthases (LasI and RhlI, respectively), and the Pseudomonas quinolone signal (PQS), which binds to PqsR, a transcription factor unrelated to LuxR-type receptors (Welsh and Blackwell, 2016a, b).…”

“…1A] and N -butyl HL, BHL) produced by LuxI-type synthases (LasI and RhlI, respectively), and the Pseudomonas quinolone signal (PQS), which binds to PqsR, a transcription factor unrelated to LuxR-type receptors (Welsh and Blackwell, 2016a, b). It has been proposed that targeting LasR may have the largest impact on QS-related virulence in P. aeruginosa (Galloway et al, 2012), since LasR activation directly upregulates certain virulence phenotypes (e.g., proteases, biofilm) and indirectly upregulates other virulence phenotypes (e.g., pyocyanin, rhamnolipid) through positive regulation of both the RhlR and PqsR systems (Asfahl and Schuster, 2018; Welsh and Blackwell, 2016a, b). Therefore, considerable efforts have been directed toward designing molecules to antagonize LasR and thereby block its associated virulence phenotypes, with notable contributions from the Bassler (O’Loughlin et al, 2013), Blackwell (Gerdt et al, 2014; Geske et al, 2007; Moore et al, 2015; O’Reilly and Blackwell, 2016), Greenberg (Muh et al, 2006; Müh et al, 2006), Meijler (Amara et al, 2011; Amara et al, 2009), Spring (Galloway et al, 2011; Hodgkinson et al, 2012), and Suga (Smith et al, 2003a; Smith et al, 2003b) laboratories.…”

Effects of Sulfide Flavors on AHL‐Mediated Quorum Sensing and Biofilm Formation of Hafnia alvei

“…Inhibition of AHL production in the presence of DADS and MMFDS may be due to the suppression of luxR expression caused by luxI , either at the transcription or translation level. Previous study has been demonstrated that both AHL and LasR are essential for achieving maximal activation of the lasB gene (Asfahl & Schuster, ).…”

“…10, 2018 r Journal of Food Science 2555 translation level. Previous study has been demonstrated that both AHL and LasR are essential for achieving maximal activation of the lasB gene (Asfahl & Schuster, 2017).…”

Focusing quorum sensing signalling by nano‐magnetic assembly

“…That is, while cell density or even cell distance have provided the context for QS initiation, it is more likely the autoinducer concentration proximal to a QS-responding cell that is the predominant factor . Indeed, the contextualization of QS activity, particularly its contributions to multicellularity, has been the motivation of several hypotheses including diffusion (Redfield, 2002) or efficiency (Hense et al, 2007) sensing, as well as attributions relative to the 'social good' (Parsek and Greenberg, 2005;Sandoz et al, 2007;Dandekar et al, 2012;Asfahl and Schuster, 2018).…”