Cytotoxic alkyl-quinolones mediate surface-induced virulence in Pseudomonas aeruginosa

Abstract

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the downstream toxins that mediate surface-dependent P. aeruginosa virulence remain unclear, as do the signaling pathways that lead to their activation. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and act directly on host cells to cause cytotoxicity. Surface-induced AQ cytotoxicity is independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. We further show that packaging of AQs in outer-membrane vesicles (OMVs) increases their cytotoxicity to host cells but not their ability to stimulate downstream quorum sensing pathways in bacteria. OMVs lacking AQs are significantly less cytotoxic, suggesting these molecules play a role in OMV cytotoxicity, in addition to their previously characterized role in OMV biogenesis. AQ reporters also enabled us to dissect the signal transduction pathways downstream of the two known regulators of surface-dependent virulence, the quorum sensing receptor, LasR, and the putative mechanosensor, PilY1. Specifically, we show that PilY1 regulates surfaceinduced AQ production by repressing the AlgR-AlgZ two-component system. AlgR then induces RhlR, which can induce the AQ biosynthesis operon under specific conditions. These findings collectively suggest that the induction of AQs upon surface association is both necessary and sufficient to explain surface-induced P. aeruginosa virulence.

Sub-Inhibitory Antibiotic Exposure and Virulence in Pseudomonas aeruginosa

“…OMVs facilitate the delivery of several virulence factors simultaneously, directly into the cytoplasm of host cells [41,42]. Such virulence factors include alkaline phosphatase, haemolytic phospholipase C, the toxin Cif, and cytotoxic alkyl quinolones [41,46]. Likewise, alkyl quinolones acting as QS molecules are also trafficked between bacteria in OMVs [47,48].…”

“…The mechanism by which surface-attached cells induce virulence towards host cells is thought to be multifactorial [46,64]. LasR is an important regulator of surface-attached virulence: upon surface attachment, lasR expression is up-regulated and the induction of LasR targets in response to the las QSSM, 3oC12-HSL, is stronger than in planktonic bacteria [65].…”

“…LasR is an important regulator of surface-attached virulence: upon surface attachment, lasR expression is up-regulated and the induction of LasR targets in response to the las QSSM, 3oC12-HSL, is stronger than in planktonic bacteria [65]. Although a full understanding of the mechanisms by which P. aeruginosa senses the presence of a surface and induces virulence is yet to be determined, flagella, type IV pili, the cell surface adhesin PilY1, and minor pili have all been identified as playing a role [46,63,64,66,67]. PilY1, which is up-regulated upon surface contact, seems to be important for initiating acute, cytotoxic virulence in surface-attached cells (as does LasR) [46,64].…”

Inhibiting quinolone biosynthesis of Burkholderia

“…10 Although the exact roles of MAQs in the Burkholderia cepacia complex still need to be investigated, the homologous AQs of P. aeruginosa indicate possible roles in suppressing the host immune response, mediating the killing of host cells and modulating interspecies and interkingdom interactions. [22][23][24]…”

“…2a). We synthesized several a-chloroketones (1-4) using the Houben-Hoesch reaction (Scheme 1) and combined them with commercially available compounds (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) to generate a small library (Fig. 2b).…”