It has been suggested for microbial pathogens that colonize the mucosal surface of a healthy host that symbiosis can be viewed as a form of molecular détente, a settled negotiation that is sustained by an ongoing chemical dialogue between the host and its flora [1
]. Even for an opportunistic pathogen, virulence expression against its host presents a fundamental tradeoff in that it will provoke immune retaliation and deplete the host of critical resources, and, as such, bacteria are constantly assessing the costs versus benefits of expressing virulence. Although bacteria use complex systems of communication like the quorum sensing signaling (QS) system to collect, process, and share information about the chemical composition of their environment [2
], whether such events are influenced by specific host-derived signals that indicate a major change in host health status is less well defined.
Our laboratory has been interested in host-derived bacterial signaling compounds that are proximate causes of microbial virulence activation during physiologic stress. To date, several host-derived bacterial signaling compounds have been identified that include adaptive elements of the immune system such as interferon γ [3
], tumor necrosis factor α [4
], and interleukin-1 [5
], as well as innate elements including adenosine [6
], epinephrine [7
], and antimicrobial peptides [11
]. While in vitro exposure to various host compounds can activate the virulence of bacteria, much remains to be learned about how these compounds are collected, processed, and transduced within the various virulence regulatory systems of bacteria.
One of the best studied systems of virulence regulation in bacteria is the QS system. The QS system functions via autoinducer molecules that are released and taken up by bacteria to provide a cell–cell communication network whereby complex assemblage behavior can be carried out by large populations of bacteria responding to local concentrations of QS molecules [2
]. In some cases, host-derived bacterial signaling molecules such as epinephrine have been shown to act as a surrogate QS autoinducer molecule [8
], activating various virulence genes in intestinal bacteria such as Escherichia coli
. In other cases, the QS system is activated by the binding of host-derived bacterial signaling molecules to specific membrane receptors on the bacteria, such as when interferon γ binds to the OprF outer membrane protein in Pseudomonas aeruginosa
We considered that opioids might function as host-derived bacterial signaling molecules given that endogenous opioids are broadly distributed within the richly innervated intestinal mucosa and exert multiple effects during stress in neuronal, immune, and intestinal epithelial cells [14
]. The intestinal tract represents a unique intersection of opioids and bacteria given the high abundance of peripheral neurons, immune cells, and bacteria in this site. Three main families of opioids have been identified based on their affinity to δ-, μ-, and κ-opioid receptors [14
] that include the endogenous opioids β-endorphin, enkephalin, and dynorphin [18
]. Following stress, endogenous opioids have been shown to act as paracrine and autocrine signals with high levels of functional redundancy and pleiotropy [21
]. These observations, coupled with the findings that neutrophils themselves can synthesize and release opioids at sites of inflammation, strongly suggest that bacteria are exposed to opioids during the course of infection.
We have been interested in the mechanism by which the human opportunistic pathogen P. aeruginosa
is activated to express a virulent phenotype during stress. We have previously shown in mice that stress results in the release of soluble compounds into the intestinal lumen that directly activate the virulence of P. aeruginosa
to disrupt the intestinal epithelial barrier [22
]. Given the abundance of neurons and immune cells in the gut that could produce opioids, we exposed strains of P. aeruginosa
to various opioids with specificity to μ-, λ-, and κ-opioid receptors and used pyocyanin (PCN) production as a biologic readout for virulence expression. Results demonstrated that only the κ-opioid receptor agonist U-50,488 induced PCN production in a dose-dependent manner. Next, we examined the effect of dynorphin, a naturally occurring κ-opioid peptide known to be present in the mammalian intestine, on its ability to produce PCN in P. aeruginosa,
and found that dynorphin potently induced PCN production. In an in vivo stress model in mice, we demonstrated that dynorphin is released into the intestinal lumen and binds to desquamated epithelia and intestinal P. aeruginosa.
Dynorphin was found to penetrate the bacterial membrane and directly induce the expression of the multiple virulence factor regulator (MvfR)–regulated operon pqsABCDE,
resulting in enhanced production of three known intercellular QS-related signals, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), 4-hydroxy-2-heptylquinoline (HHQ), and Pseudomonas
quinolone signal (3,4-dihydroxy-2-heptylquinoline [PQS]) [23
]. Exposure of P. aeruginosa
strain PAO1 to κ-opioid receptor agonists U-50,488 and dynorphin resulted in enhanced virulence as judged by suppressed growth of the probiotic microorganisms Lactobacillus
spp. and the nematode Caenorhabditis elegans
. Taken together, these studies provide novel insight into the mechanism by which P. aeruginosa
is activated to express virulence in response to host stress by processing the opioid peptide dynorphin into its QS circuitry.