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, 6 (3), e1000810

Homeostatic Interplay Between Bacterial Cell-Cell Signaling and Iron in Virulence


Homeostatic Interplay Between Bacterial Cell-Cell Signaling and Iron in Virulence

Ronen Hazan et al. PLoS Pathog.


Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. PqsE, a key mediator of the MvfR regulon activation, functions independently of AA and its derivatives.
(A and B) Pyocyanin production was measured from PA14 and mutants with and without constitutive expression of PqsE or MvfR as a consequence of the presence of pDN19pqsE or pDN18mvfR plasmids, respectively. (A) AA is a triple mutant with non-functional phnAB, trpE and kynBU that does not produce anthranilate. Production of pyocyanin (+ Phz) was achieved by co-culturing two sets of cells one constitutively expressing phzA2-G2, and the other phzM and phzS genes encoding the phenazines and pyocyanin biosynthetic genes respectively. Asterisks in A show strains harboring the plasmid pDN19pqsE that are significantly different (P value <0.01) from PA14 harboring that plasmid. (C–D) PqsE is essential for the virulence of P. aeruginosa against Cryptococcus neoformans independently of HAQs. PqsE was constitutively expressed in mvfR mutant cells. An empty vector served as a control (−). (C) 1 µL of bacterial culture was spotted onto YPD top-agar where yeast cells were plated. Yeast killing zones were formed only around the PA14 and mutant cells expressing PqsE. (D) The death of yeast cells within the killing zone was demonstrated by assessing their viability on YPD plates. (E) PqsE causes fly mortality in absence of HAQs. Survival kinetics of Drosophila melanogaster was assessed using a fly feeding assay. The survival kinetics of pqsA and pqsE infected flies was significant different (P value <0.005) form that of PA14-infected flies. However, the kinetics of pqsA + PqsE- infected flies did not differ significantly from that of the PA14-infected flies(P value = 0.27).
Figure 2
Figure 2. The homeostatic regulation of the signaling molecules HHQ and PQS is orchestrated by PqsE.
Effect of PqsE on pqs operon gene expression, and production of HAQs and AA. (A) Fold change in expression of phn and pqs operons in pqsE mutant and PA14 constitutively expressing PqsE versus PA14. (B) GFP intensity derived from a pqsA-GFP(ASV) reporter fusion; (C) HAQs and (D) AA levels as assessed by LC-MS. t-tests (p = 0.001 for HHQ and p = 0.004 for PQS) showed that the difference between PA14 and PA14+PqsE is statistically significant.
Figure 3
Figure 3. MvfR network regulation requires finely tuned cooperation between the MvfR component PqsE and the AHL QS regulator RhlR.
(A) The expression of pqsA was determined by measuring GFP emission. A pqsA-GFP (ASV) fusion in the rhlR mutant harboring pDN19pqsE was used to determine pqsA expression levels. (B) Pyocyanin levels were measured from various PA14 mutants harboring either pDN19pqsE or pUCP20rhlR plasmids. Empty vector served as control.
Figure 4
Figure 4. Negative homeostatic feedback regulation on MvfR regulon products and activity is mediated via cell-cell signaling molecule concentration.
(A) Pyocyanin levels were assessed in PA14 and mutants cells harboring the plasmid pDN19pqsE with or without the addition of PQS or HHQ (20 mg/L). t-tests (p<0.05) showed that the difference between untreated and PQS/HHQ treated cells was statistically significant (B–C) Pyocyanin levels were determined following the addition of PQS over a broad-range of concentrations using a PQS non-producing strain (B) or using a narrow range of PQS concentrations in PQS-producing strains (C). PqsE was constitutively expressed (+PqsE). The empty vector was used as a control. phz genes were expressed following co-culture of pqsE cells constitutively expressing phzA2-G2 with pqsE cells constitutively expressing the phzM and phzS genes. The cells were grown in the presence of exogenously added PQS and pyocyanin production measured by measuring the OD600 nm. (D) The expression of pqsA was determined using a pqsA-GFP (ASV) fusion in a pqsA-::pqsH double mutant in the presence of various concentrations of HHQ and PQS. (E) A Venn diagram showing the number of PqsE-regulated genes counterbalanced by PQS. The data was adapted from Table S1.
Figure 5
Figure 5. Homeostatic interplay between PQS and iron: Iron fine-tunes PQS activities.
The effect of iron on MvfR induction was tested using the pqsA-GFP reporter in PA14 (A) and PA14 pqsA ::pqsH cells treated with PQS (1 mg/L) (B). The effect of iron on pyocyanin production was tested when PQS was supplied at 1 mg/L or 20 mg/L (C). The cells were grown in low iron medium D-TSB or in media supplemented with iron (FeCl3 or FeSO4, 200 µM). Asterisks show samples that are statistically significant different (P value<0.05) from the PQS 1 mg/L treated sample.
Figure 6
Figure 6. Schematic of the positive and negative homeostatic interplay among the MvfR regulon components PqsE, and PQS and HHQ with RhlR and iron.
PqsE (green), HHQ and PQS (blue) and iron (red) play a dual role in up- or down-regulating the MvfR regulon. The outcome—that is the level of downstream gene expression translated into the bacterial virulence response—is the integrated sum of these interactions. Positive loops (thin lines): (1) MvfR is induced by HHQ and its derivative PQS to express phn and pqs operons, which are in turn (2) responsible for the synthesis of HAQs. PqsE is not required for HAQ synthesis and does not need AA or its derivatives for its “bottleneck” function, (3) controlling the expression of many virulence factors in cooperation with the AHL regulator RhlR. (4) PqsE also controls many iron starvation response genes, such as PvdS and siderophores. (5) PvdS in turn up-regulates the transcription of mvfR via an iron starvation box. (6) Low iron conditions also contribute to the induction of PvdS and other iron-related regulators to activate the iron response including (7) uptake of iron into the cell by siderophores as well as (8) induction of the virulence response. Negative loops (thick lines): (9) PqsE in cooperation with RhlR down-regulates the expression of the phn and pqs operons, thus reducing HAQ production. When a threshold concentration of HHQ is reached, (10) HHQ down-regulates the pqs operon. (11) PQS at high physiological levels in turn counterbalances the expression of PqsE-controlled genes, including many virulence factors. High levels of iron in presence of low levels of PQS, reduce P. aeruginosa virulence, at least in part, by (12) binding and inactivating PQS. In contrast, when PQS is at high physiological levels its inactivation by iron will increase virulence by reducing the negative PQS counterbalance and thus sustain the positive loops that include (13) iron starvation as a result of PQS trapping iron. (14) The integration of these processes enforces a fine-tuning of MvfR regulon gene expression levels, therefore determining the magnitude of virulence.

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