Interkingdom signaling between Pseudomonas aeruginosa and mammalian cells

Date

2005-12

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Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that commonly infects immunocompromised patients. P. aeruginosa utilizes a cell densitydependent signaling phenomenon known as quorum sensing (QS) to coordinate the expression of virulence factors during infection. QS involves two components, an autoinducer (AI) synthase protein that produces AI and a transcriptional activator protein from the LuxR family, which is activated and promotes the expression of QS target genes when it is bound to cognate AI. One AI produced by P.aeruginosa, N-30-dodecanoylhomoserine lactone (30-C12), has direct effects mammalian host cells. We hypothesize that 30-C12 directly modulates host cell responses by activating signaling transduction pathways. We determined that P. aeruginosa Ais can enter and function in mammalian cells by expressing AI-detector proteins in mammalian cells. AI-detector proteins were generated by fusing the bacterial AI receptor proteins to a transcriptional activation domain and a nuclear localization signal so that they would function as activatable transcription factors in mammalian cells. Here we also report that treatment of murine fibroblasts with 30-C12 results in an increase in the cytoplasmic free calcium concentration. Our studies indicated that calcium is released from endoplasmic reticulum (ER) stores through the generation of inositol (1,4,5)-triphosphate (IP3) by phospholipase C and subsequent opening of IP3 receptor calcium channels in the ER membrane. With the application of calcium signaling inhibitors, we demonstrated that 30-C12-activated calcium signaling in fibroblasts was linked to the induction of apoptosis but not to proinflammotory responses, which are both activated in fibroblasts treated with 30-Cl2. We explored the potential for the AI binding domain (AIBD) ofLuxR family members to serve as transferable modules that could render a protein under inducible control. To test this, we created fusion proteins from the LuxR homologues of P. aeruginosa, LasR and RhlR, and a LuxR homologue expressed in the soil bacterium Agrobacterium tumefaciens (TraR). We fused the AIBD from LasR, RhlR, and TraR to a potent synthetictranscription factor, Gal4-VP16. We obseived that the addition of each AIBD to Gal4- VP 16 inhibited its activity, but only the AIBD from LasR was activated upon the addition of cognate autoinducer.

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Keywords

Pseudomonas aeruginosa, Mammalian cells, Interkingdom signaling, Microbiology

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