Intercellular communication in Helicobacter pylori: luxS is essential for the production of an extracellular signaling molecule - PubMed (original) (raw)

Comparative Study

Intercellular communication in Helicobacter pylori: luxS is essential for the production of an extracellular signaling molecule

M H Forsyth et al. Infect Immun. 2000 Jun.

Abstract

Individual bacteria of numerous species can communicate and coordinate their actions via the production, release, and detection of extracellular signaling molecules. In this study, we used the Vibrio harveyi luminescence bioassay to determine whether Helicobacter pylori produces such a factor. Cell-free conditioned media from H. pylori strains 60190 and 26695 each induced >100-fold-greater luminescence in V. harveyi than did sterile culture medium. The H. pylori signaling molecule had a molecular mass of <10 kDa, and its activity was unaffected by heating to 80 degrees C for 5 min or protease treatment. The genome sequence of H. pylori 26695 does not contain any gene predicted to encode an acyl homoserine lactone synthase but does contain an orthologue of luxS, which is required for production of autoinducer-2 (AI-2) in V. harveyi. To evaluate the role of luxS in H. pylori, we constructed luxS null mutants derived from H. pylori 60190 and 26695. Conditioned media from the wild-type H. pylori strains induced >100-fold-greater luminescence in the V. harveyi bioassay than did conditioned medium from either mutant strain. Production of the signaling molecule was restored in an H. pylori luxS null mutant strain by complementation with a single intact copy of luxS placed in a heterologous site on the chromosome. In addition, Escherichia coli DH5alpha produced autoinducer activity following the introduction of an intact copy of luxS from H. pylori. Production of the signaling molecule by H. pylori was growth phase dependent, with maximal production occurring in the mid-exponential phase of growth. Transcription of H. pylori vacA also was growth phase dependent, but this phenomenon was not dependent on luxS activity. These data indicate that H. pylori produces an extracellular signaling molecule related to AI-2 from V. harveyi. We speculate that this signaling molecule may play a role in regulating H. pylori gene expression.

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Figures

FIG. 1

Production of an extracellular signaling molecule by H. pylori. V. harveyi BB170 was inoculated into AB medium containing 10% CM from V. harveyi BB152, 10% CM from H. pylori 26695, or 10% sterile brucella broth. Cultures were incubated at 25°C with constant agitation, and aliquots were removed at serial time points for measurement of luminescence (A) and viable cell count (B). Luminescence is expressed in relative light units (luminescence per 106 viable V. harveyi BB170 cells). At the 5-h time point, CM from V. harveyi BB152 and H. pylori 26695 induced >100-fold-greater luminescence than did sterile brucella broth.

FIG. 2

Alignment of the deduced H. pylori LuxS sequence with deduced LuxS sequences from four other bacterial species. LuxS sequences from H. pylori 26695 (GenBank accession no. AE000532), S. aureus (preliminary sequence data obtained from The Institute for Genomic Research website at

http://www.tigr.org/

), B. subtilis (accession no. Z9919), C. perfringens (accession no. AB028629), and V. harveyi (accession no. AAD 17292) were aligned using the ClustalW algorithm. H. pylori LuxS is most closely related to LuxS from S. aureus (67% amino acid identity; 15% similarity). Positions of amino acid identity are indicated by asterisks.

FIG. 3

Role of H. pylori luxS in the production of an extracellular signaling molecule. (A) H. pylori L60-1 is a luxS null mutant strain in which luxS has been disrupted by the insertion of a CAT cassette. H. pylori L60-2 contains the same luxS mutation as in L60-1, but an intact copy of luxS has been inserted within the ureA gene. (B) V. harveyi BB170 was inoculated into AB medium containing 10% CM from H. pylori 60190 (wild type), 10% CM from H. pylori L60-1 (60190 luxS::CAT), 10% CM from H. pylori L60-2 (60190 luxS::CAT ureA::luxS-aphA3), or 10% brucella broth. Comparison of V. harveyi luminescence values at the 5-h time point demonstrates that wild-type H. pylori 60190 CM induces 100-fold-greater luminescence than does either H. pylori L60-1 CM or the brucella broth control. At the 5-h time point, CM from the _luxS_-complemented H. pylori strain, L60-2, induces approximately 100-fold-greater luminescence than does CM from H. pylori L60-1.

FIG. 4

Kinetics of H. pylori signaling molecule production. H. pylori 60190 was cultured in brucella broth containing 5% FBS, and aliquots were removed at serial time points for measurement of cell density (OD600) (asterisks) and the capacity of CM to induce luminescence in the V. harveyi BB170 bioassay system at the 5-h time point (open bars). Luminescence induction is reported as a ratio of relative light units in the presence of H. pylori 60190 CM compared to relative light units in the presence of uninoculated brucella broth. The results shown here are representative of results obtained in three independent experiments.

FIG. 5

Growth phase regulation of H. pylori vacA transcription. H. pylori 60190 VX-1 (containing a vacA::xylE transcriptional fusion) and H. pylori VX-1/LC-1 (containing both a vacA::xylE transcriptional fusion and a luxS::CAT mutation) were grown in brucella broth containing 5% FBS. Aliquots were removed at serial time points for measurement of XylE specific activity (A) and cell density (OD600) (B). Levels of vacA transcription, as evidenced by XylE activities, were growth phase dependent, but this phenomenon was not dependent on LuxS function.

FIG. 6

H. pylori luxS complements the luxS frameshift mutation of E. coli DH5α. E. coli DH5α, which bears a nonfunctional luxS (44), was transformed with a plasmid containing the intact H. pylori luxS gene (pLuxS), a plasmid containing a disrupted H. pylori luxS gene (pLuxS::CAT), or the cloning vector alone (pGEM-T). Each strain was grown in brucella broth to mid-logarithmic phase (OD600 of ∼0.5), and cell-free CM were prepared. The CM were tested for the capacity to induce luminescence in the V. harveyi bioassay at the 5-h time point. Sterile, pristine brucella broth was used as a control, and all induction values are relative to this sample. CM from V. harveyi BB152 was used as a positive control for the bioassay. The DH5α strain bearing a functional H. pylori luxS (pLuxS) induced high levels of luminescence in V. harveyi BB170. In contrast, DH5α strains bearing either a nonfunctional _luxS_Hp (pLuxS::CAT) or the vector alone were incapable of inducing luminescence in this bioassay. Results shown are representative of three independent experiments.

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Intercellular communication in Helicobacter pylori: luxS is essential for the production of an extracellular signaling molecule - PubMed (original) (raw)