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 surface-induced 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.
Highlights
The opportunistic human pathogen P. aeruginosa infects a wide range of hosts such as mammals, plants, insects, and fungi [1], and is a major contributor to the morbidity of cystic fibrosis patients [2] and hospital-acquired infections [3]
Given the multiple activities involved in pathogenesis, we recently developed a quantitative imaging-based host cell killing assay to study the factors acutely required for killing host cells during short timescales [5]
Since well-characterized cytotoxins such as T3SS and Vfr targets are not necessary for surface-induced host-cell killing in this assay, we sought to address the outstanding questions of which specific toxins mediate host cell killing in response to surface attachment
Summary
The opportunistic human pathogen P. aeruginosa infects a wide range of hosts such as mammals, plants, insects, and fungi [1], and is a major contributor to the morbidity of cystic fibrosis patients [2] and hospital-acquired infections [3]. Given the multiple activities involved in pathogenesis, we recently developed a quantitative imaging-based host cell killing assay to study the factors acutely required for killing host cells during short timescales [5]. This assay revealed that cytotoxicity is activated by attachment of P. aeruginosa to a solid surface [5]. This surface-induced cytotoxicity does not require the Type-IV Pilus (TFP), TFP-associated signaling (PilA-ChpVfr/cAMP), or Type III Secretion Systems (T3SS), but does require two regulatory proteins, LasR and PilY1 [5].
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