Diethyl Phthalate, a Chemotactic Factor Secreted by Helicobacter pylori

Abstract

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37°C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.

Conditioned Medium from EnterohemorrhagicEscherichia coli-Infected T84 Cells Inhibits Signal Transducer and Activator of Transcription 1 Activation by Gamma Interferon

“…Furthermore, this factor may not be a peptide, since its ability to suppress Stat1 activation was resistant to treatment with proteinase K, trypsin, heat inactivation, and sodium orthovanadate. Indeed, bacteria can secrete nonpeptide factors that interact with host cells, such as the monocyte chemoattractant diethyl phthalate by Helicobacter pylori (27). However, since peptides can be heat inactivation resistant (16), and since protease treatment may leave behind an active, smaller peptide, the exact molecular identity of the factor(s) requires further determination.…”

“…Also, proteasome inhibition can prevent NF-B activation, and NF-B activity is implicated in suppression of Stat1 activation (27) and SOCS expression (32). Preincubation with the proteasome inhibitor MG-132 did not reverse the ability of EHEC to suppress IFN-␥-induced Stat1 tyrosine phosphorylation, indicating that the effect is proteasome-independent (Table 1).…”

Basophils Infiltrate Human Gastric Mucosa at Sites of Helicobacter pylori Infection, and Exhibit Chemotaxis in Response to H. pylori-derived Peptide Hp(2–20)

“…Checkboard analyses were performed to discriminate between chemotaxis and nondirected migration (chemokinesis) of basophils. In these experiments, basophils were placed in the upper chemotactic chambers and various concentrations of Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), FMLP, or PACGM buffer were added to the upper or lower wells or to both. Spontaneous migration (chemokinesis) was determined in the absence of chemoattractant or when stimuli were added to either the lower or upper chambers.…”

“…A cecropin-like peptide, Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), with a sequence (NH 2 -AKKVFKRLE KLFSKIQNDK-COOH) identical with that of the amino-terminal part of ribosomal protein L1 in H. pylori (50), was synthesized and purified by Innovagen (Lund, Sweden). The peptide, Hp1, with a sequence (NH 2 -AKKVFKRLELLFSKIQNDK-COOH), in which proline in position 10 had been substituted with lysine, and the hexapeptide Trp-Lys-Tyr-MetVal-D-Met-NH 2 (WKYMVm) was synthesized by Innovagen.…”

“…Many chemotactic factors secreted by H. pylori have been identified (10 -12). Recently, a cecropin-like H. pylori peptide (Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)) was found to be a monocyte chemoattractant (11). Cecropins are a group of peptides first discovered in the context of insect immunity and subsequently identified in bacteria (13).…”