We report that 2 g of azithromycin/ml inhibits the quorum-sensing circuitry of Pseudomonas aeruginosa strain PAO1. Addition of synthetic autoinducers partially restored the expression of the trancriptional activator-encoding genes lasR and rhlR but not that of the autoinducer synthase-encoding gene lasI. We propose that azithromycin interferes with the synthesis of autoinducers, by an unknown mechanism, leading to a reduction of virulence factor production.

During nutrient starvation, Escherichia coli elicits a stringent response involving the ribosome-associated protein RelA. Activation of RelA results in a global change in the cellular metabolism including enhanced expression of the stationary-phase sigma factor RpoS. In the human pathogen Pseudomonas aeruginosa, a complex quorum-sensing circuitry, linked to RpoS expression, is required for cell density-dependent production of many secreted virulence factors, including LasB elastase. Quorum sensing relies on the activation of specific transcriptional regulators (LasR and RhlR) by their corresponding autoinducers (3-oxo-C 12 -homoserine lactone [HSL] and C 4 -HSL), which function as intercellular signals. We found that overexpression of relA activated the expression of rpoS in P. aeruginosa and led to premature, cell density-independent LasB elastase production. We therefore investigated the effects of the stringent response on quorum sensing. Both lasR and rhlR gene expression and autoinducer synthesis were prematurely activated during the stringent response induced by overexpression of relA. Premature expression of lasR and rhlR was also observed when relA was overexpressed in a PAO1 rpoS mutant. The stringent response induced by the amino acid analogue serine hydroxamate (SHX) also led to premature production of the 3-oxo-C 12 -HSL autoinducer. This response to SHX was absent in a PAO1 relA mutant. These findings suggest that the stringent response can activate the two quorum-sensing systems of P. aeruginosa independently of cell density.

Pseudomonas aeruginosa controls the secretion of extracellular virulence factors, including rhamnolipids and LasB elastase, by the las and rhl quorum-sensing systems. Here, we mutated the dksA gene of P. aeruginosa by insertion of an ⍀-Hg cassette. The mutant displayed growth rates similar to that of the wild type in rich medium but was impaired in growth in defined minimal medium. Production of rhamnolipids and LasB elastase by the dksA mutant was only 4 and 10%, respectively, of wild-type levels. These defects could be partially complemented by introduction of the plasmid-encoded dksA genes from P. aeruginosa or Escherichia coli. In the dksA mutant, the expression of rhlI was increased early during exponential growth, but expression of other quorum-sensing regulator genes-lasR, lasI, and rhlR-was not affected. Although the transcription of the lasB and rhlAB genes was comparable between the dksA mutant and the wild-type strain in peptone tryptic soy broth medium, we observed reduced translation of both genes in the dksA mutant. Similarly, we found that full translation of lasB and rhlAB genes in E. coli also requires the dksA gene. DksA is therefore a novel regulator involved in the posttranscriptional control of extracellular virulence factor production in P. aeruginosa.The opportunistic pathogen Pseudomonas aeruginosa is responsible for severe nosocomial infections in immunocompromised and intubated patients (4). In addition, P. aeruginosa is the most commonly found pathogen in cystic fibrosis (CF) patients and is responsible for progressive lung tissue destruction leading to respiratory failure (4). P. aeruginosa produces a wide spectrum of secreted virulence factors, including LasB elastase, rhamnolipids, pyocyanin, lipase, and hydrogen cyanide (30). The las and rhl quorum-sensing systems regulate the production of these factors in a cell density-dependent manner. This regulation relies on the accumulation in the medium of two autoinducer (AI) molecules, 3-oxo-C 12 -homoserine lactone (3-oxo-C 12 -HSL) and C 4 -HSL, that induce the las and rhl quorum-sensing systems when the bacterial cell density reaches a certain threshold (quorum), leading to transcription of specific genes and the production of the virulence factors cited above. Both systems involve a transcriptional regulator (RhlR and LasR, respectively) and an AI synthase (RhlI and LasI, respectively) (30). A hierarchy was proposed in which the las system activates the rhl system by inducing the transcription of the activator gene rhlR (12,20).Additional layers of regulation of the quorum-sensing circuitry have been described. These include the transcriptional regulator vfr (1), a homologue of the Escherichia coli cyclic AMP-binding protein, the GacA two-component regulator (23), the enzyme polyphosphate kinase (22), and the mvaT gene product which modulates the timing of quorum-sensing activation (5). Recently, we have shown that overexpression of the starvation response regulator encoded by relA, leading to increased production of the nutrient str...

Myeloid differentiation factor-2 (MD-2) is a lipopolysaccharide (LPS)- IntroductionRecognition of bacteria and bacterial molecules is a key step in the initiation of an innate immune response in animals and in humans. Several host humoral and cell-surface proteins are implicated in the innate immune recognition of endotoxin (lipopolysaccharide, LPS), for example. These include LPS-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4), and the myeloid differentiation factor-2 (MD-2). [1][2][3][4] Mice deficient in any one of these proteins display a similar hyporesponsiveness to LPS challenge. [5][6][7][8] MD-2 is a glycoprotein of approximately 17-25 kDa coexpressed with TLR4 at the surface of various cell types, principally those of the myeloid and endothelial lineages. 9,10 MD-2 mRNA is found in a variety of tissues. 11 Despite of the absence of a transmembrane domain, it is found attached to the cell surface via its interaction with TLR4 through specific and recently identified epitopes. 2,9,12,13 The sequence events by which LPS interacts with the various proteins of its receptor complex has recently been unravelled. LPS first binds CD14, which transfers it to a hydrophobic pocket within the MD-2 glycoprotein, resulting in TLR4-dependent activation of cells. 5,[13][14][15] MD-2 also exists as a soluble protein (sMD-2) secreted by various cell types, mainly as a large disulfide-bound multimeric glycoprotein but also as oligomers and monomers. 9,16,17 However, only sMD-2 monomers seem to bind to LPS and confer responsiveness to TLR4 expressing cells. 18,19 Little is known about the sMD-2 secretory pathway. MD-2 possesses a consensus sequence for a secretory signal peptide consisting of a 16-amino-acid NH 2 -terminal hydrophobic stretch. 2 An alternatively spliced MD-2 variant inhibiting TLR4 expression has been described in mice but not in humans. 20 Soluble proteins are essential in the host response to microbial infections. Some of these proteins, such as LBP and soluble CD14, are cofactors for the activation of cells by bacterial molecules. 3,21 Others function as opsonins (LBP, serum amyloid protein A, complement, and mannose-binding lectins), or are directly bactericidal, such as the bactericidal permeability-increasing (BPI) protein and complement, for example. [22][23][24][25][26] It is noteworthy that many of these proteins are acute-phase proteins. 24,[27][28][29][30][31] We have recently reported a marked increase in sMD-2 activity in plasma from patients with sepsis, and, to a lesser extent, in plasma from patients with an acute exacerbation of rheumatoid arthritis. 32 Increased levels of sMD-2 in septic plasma were subsequently confirmed. 33 sMD-2 has also been detected in other body fluids, including urine and lung edema fluid from patients with sepsis. 32 It has been proposed that high concentrations of sMD-2, like soluble CD14 and high-density lipoproteins, may act as a sink for LPS. 34 The relative concentration of monomer versus polymer in human body fluids is unknown. The origin and the re...