Soluble MD-2 is an acute-phase protein and an opsonin for Gram-negative bacteria.

Myeloid differentiation factor-2 (MD-2) is a lipopolysaccharide (LPS)-binding protein usually coexpressed with and binding to Toll-like receptor 4 (TLR4), conferring LPS responsiveness of immune cells. MD-2 is also found as a soluble protein. Soluble MD-2 (sMD-2) levels are markedly elevated in plasma from patients with severe infections, and in other fluids from inflamed tissues. We show that sMD-2 is a type II acute-phase protein. Soluble MD-2 mRNA and protein levels are up-regulated in mouse liver after the induction of an acute-phase response. It is secreted by human hepatocytic cells and up-regulated by interleukin-6. Soluble MD-2 binds to Gram-negative but not Gram-positive bacteria, and sMD-2 secreted by hepatocytic cells is an essential cofactor for the activation of TLR4-expressing cells by Gram-negative bacteria. Soluble MD-2 opsonization of Gram-negative bacteria accelerates and enhances phagocytosis, principally by polymorphonuclear neutrophils. In summary, our results demonstrate that sMD-2 is a newly recognized type II acute-phase reactant, an opsonin for Gram-negative bacteria, and a cofactor essential for the activation of TLR4-expressing cells. This suggests that sMD-2 plays a key role in the host innate immune response to Gram-negative infections.

Cyclic stretch of human lung cells induces an acidification and promotes bacterial growth.

The reasons for bacterial proliferation in the lungs of mechanically ventilated patients are poorly understood. We hypothesized that prolonged cyclic stretch of lung cells influenced bacterial growth. Human alveolar type II-like A549 cells were submitted in vitro to prolonged cyclic stretch. Bacteria were cultured in conditioned supernatants from cells submitted to stretch and from control static cells. Escherichia coli had a marked growth advantage in conditioned supernatants from stretched A549 cells, but also from stretched human bronchial BEAS-2B cells, human MRC-5 fibroblasts, and murine RAW 264.7 macrophages. Stretched cells compared with control static cells acidified the milieu by producing increased amounts of lactic acid. Alkalinization of supernatants from stretched cells blocked E. coli growth. In contrast, acidification of supernatants from control cells stimulated bacterial growth. The effect of various pharmacological inhibitors of metabolic pathways was tested in this system. Treatment of A549 cells and murine RAW 264.7 macrophages with the Na(+)/K(+)-ATPase pump inhibitor ouabain during cyclic stretch blocked both the acidification of the milieu and bacterial growth. Several pathogenic bacteria originating from lungs of patients with ventilator-associated pneumonia (VAP) also grow better in vitro at slightly acidic pH (pH 6-7.2), pH similar to those measured in the airways from ventilated patients. This novel metabolic pathway stimulated by cyclic stretch may represent an important pathogenic mechanism of VAP. Alkalinization of the airways may represent a promising preventive strategy in ventilated critically ill patients.

DksA represses ribosomal gene transcription in Pseudomonas aeruginosa by interacting with RNA polymerase on ribosomal promoters.

In Escherichia coli transcription of ribosomal RNA (rRNA) is regulated by the H-NS and Fis proteins, as well as by the small signal molecule ppGpp and the initiating nucleotides. During amino acid starvation, the concentration of ppGpp increases, and binding of this alarmone to RNA polymerase (RNAP) leads to inhibition of rRNA transcription, a regulatory event called stringent response. Here we show that in Pseudomonas aeruginosa DksA, a protein with pleiotropic effects, is a negative regulator of rRNA transcription both during exponential growth and stringent conditions. A dksA mutant overexpresses rRNA, without being affected in the production of ppGpp. Cell-fractionation and chromosome immunoprecipitation experiments demonstrate that DksA is associated with DNA, in particular with promoters of ribosomal genes in vivo. The binding to rRNA promoters specifically increases during stringent response, and correlates with the binding of RNAP to these regions. Moreover DksA can be copurified with RNAP subunits in vivo. DNA band shift experiments show that DksA, in synergy with ppGpp, increases the binding of RNAP to ribosomal promoters. Therefore DksA might be a new regulator of rRNA transcription in P. aeruginosa.

Posttranscriptional control of quorum-sensing-dependent virulence genes by DksA in Pseudomonas aeruginosa.

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 Omega-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.