Human β-Defensin 2 Induces Extracellular Accumulation of Adenosine in Escherichia coli

Abstract

Human ␤-defensins are host defense peptides performing antimicrobial as well as immunomodulatory functions. The present study investigated whether treatment of Escherichia coli with human ␤-defensin 2 could generate extracellular molecules of relevance for immune regulation. Mass spectrometry analysis of bacterial supernatants detected the accumulation of purine nucleosides triggered by ␤-defensin 2 treatment. Other cationic antimicrobial peptides tested presented variable outcomes with regard to extracellular adenosine accumulation; human ␤-defensin 2 was the most efficient at inducing this response. Structural and biochemical evidence indicated that a mechanism other than plain lysis was involved in the observed phenomenon. By use of isotope ( 13 C) labeling, extracellular adenosine was found to be derived from preexistent RNA, and a direct interaction between the peptide and bacterial nucleic acid was documented for the first time for ␤-defensin 2. Taken together, the data suggest that defensin activity on a bacterial target may alter local levels of adenosine, a well-known immunomodulator influencing inflammatory processes. Human ␤-defensins (hBD) are cationic antimicrobial peptides produced predominantly by epithelial cells (1). Notably, in the intestine, an environment populated by a dense and rich microbial community, epithelial defensins reinforce the host innate defense (2). These peptides are normally expressed at low basal levels, but hBD-2 and -3 can be upregulated by a variety of microbial and inflammatory stimuli (3, 4). In particular, altered hBD-2 expression has been reported to correlate with the development of intestinal inflammation (5, 6). On the other hand, intestinal inflammation has been associated with alterations in the microbiota-for example, with shifts in the relative abundance of Proteobacteria, such as Escherichia coli (7,8). Elucidating how the interaction between hBD and their bacterial targets impact inflammatory processes can aid our understanding of host-microbe networking in health and disease.The most-studied mechanism of action of cationic antimicrobial peptides involves membrane permeabilization with eventual cell lysis; however, other mechanisms and cellular targets have been described for many peptides, including human defensins (9-12). Moreover, in addition to their antimicrobial activity, manifold immunomodulatory properties of defensins have been reported, and many of these reports describe direct effects on immune cells and cytokine expression (13-15). In some cases, the interaction of peptides with bacterial components is implicated in their immunomodulatory effects (16,17). In this context, the present study hypothesized that hBD-2 activity on E. coli could result in the generation of extracellular mediators with relevance for immunomodulation. MATERIALS AND METHODSChemicals. Lyophilized synthetic human ␤-defensin 2, human ␤-defensin 3, and human ␣-defensin 5 were purchased from the Peptide Institute Inc., sheep myeloid antimicrobial peptide 29 (SMAP-29) from AnaSpe...

Release of Periplasmic Nucleotidase Induced by Human Antimicrobial Peptide in E. coli Causes Accumulation of the Immunomodulator Adenosine

“…Therefore, we have decided to use a substitute, less expensive product, sheep-myeloid antimicrobial peptide 29 (SMAP-29); this is a mammalian α-helical cathelicidin which was shown to elicit an extracellular Ado accumulation response in E . coli similar to hBD-2 [ 14 ]. SMAP-29 was used for the experiments presented in Fig 2A .…”

“…Our previous findings demonstrated that hBD-2-treated E . coli undergoes plasmolysis, releasing nucleotide-related metabolites while retaining intracellular macromolecules [ 14 ]. This scenario could account for the presence of periplasmic 5’ nucleotidase in the extracellular medium, which in turn could be the soluble bacterial factor responsible for converting AMP into Ado after bacteria had been challenged with defensin.…”

“…Detection of adenosine nucleoside and adenosine monophosphate nucleotide was performed by targeted tandem mass spectrometry (multiple reaction monitoring mode) in a 6460 TripleQuad LC-MS system (Agilent Technologies, Santa Clara, CA, USA) as described [ 14 ].…”

“…Previous work by our group has demonstrated that a non-lytic mechanism of action of human β-defensin-2 (hBD-2) can trigger membrane dissociation (plasmolysis) and extracellular accumulation of adenosine (Ado) from the bacterium Escherichia coli [ 14 ]. Given the multitude of biological activities attributed to Ado signaling during inflammation, this novel perspective placed hBD-2 as a component of the host-microbial communication arsenal, able to elicit from the bacterial target the release of an important immunomodulatory molecule.…”

Applications and impacts of stable isotope probing for analysis of microbial interactions

“…To answer the question whether adenosine is synthesized de novo at defensin stress or release from already existing RNA, cells were fed with [U-13 C]-glucose before their exposure to defensin and compared to those fed with the same labeled substrate during defensin stress. Analysis of the formed adenosine showed 13 C-enriched adenosine only from cells exposed before the defensin stress confirming that already existing but not de novo synthesized adenosine was released (Estrela et al 2013). C-RNA-separation protocols even active community members comprising only a tiny fraction of all cells can be detected.…”