P. aeruginosa produce N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a so-called “quorum-sensing molecule” that provides signals for the production of virulence factors and for bacterial biofilm formation in a paracrine manner. We now found that 3OC12-HSL, but not its 3-deoxo-isomer or acyl homoserine lactones with shorter fatty acids, is also able to activate human polymorphonuclear neutrophils (PMN) in vitro: 3OC12-HSL enhanced the phagocytosis of opsonised bacteria in vitro; up-regulated the surface expression of phagocytosis-related receptors, and was chemotactic for PMN. Because induction of chemotaxis implies the polarisation of the cell by receptors expressed on the surface, we performed uptake studies with radiolabelled 3OC12-HSL. At 4° C we found saturable binding of the radiolabelled 3OC12-HSL, which could be inhibited by an excess of unlabelled 3OC12-HSL, indicating specificity of binding, and hence expression of a distinct surface receptor. By use of selective inhibitors, a signalling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, mitogen activated protein kinase C was delineated, but in contrast to the well-studied chemokines C5a and interleukin 8, the chemotaxis in response to 3OC12-HSL did not depend on pertussis toxin-sensitive G proteins. Selective surface receptors for 3OC12-HSL have been identified in various bacteria species, but scrutinising a human gene bank did not reveal homologous structures. While the characterisation of the surface receptor awaits further studies, the functional consequence of the cross-kingdom signalling is obvious: by recognising and responding to 3OC12-HSL PMN are attracted to the site of a developing biofilm, and thus may prevent its progression and by that persistent infection.

P.aeruginosa causes acute and chronic-destructive infections, particularly wound infections, or device-associated infections by colonising respiratory tubes, catheters, or implants. The pathogenicity of P.aeruginosa is largely attributed to the relative resistance towards host defence. Especially when organised as biofilms, the bacteria evade phagocytosis and killing by polymorphonuclear neutrophils (PMN).

To elucidate the evasion mechanisms, the migration of PMN towards and through P.aeruginosa biofilms was studied. Migration of PMN towards P.aeruginosa biofilms was tested using various in vitro techniques.

We found that PMN migrated towards developing P.aeruginosa biofilms, attracted by the quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL). Mature biofilms which no longer produced 3OC12-HSL did not attract PMN. Addition of interleukin 8, a potent chemokine, restored the migratory capacity. Once arrived at the biofilms, PMN readily attached with no important difference between developing and mature biofilms. Migration into and penetration of the films, however, was only seen with developing films. By mass spectroscopy it became obvious that a major difference between developing and mature biofilms was the composition of the extracellular polymer substance, of which alginate is a prominent component. A series of experiments with isolated alginate showed that PMN did not migrate on or into alginate-containing matrices, but remained affixed at the contact site just as they did on mature biofilms. The mechanism of this firm attachment is still under investigation; prominent up-regulation of various adhesion molecules was seen, which could provide possible explanation.

Mature biofilms, most probably due to the composition of the extracellular polymer substance, do not allow the penetration of PMN. Consequently, bacteria embedded in deeper layers of the biofilm are protected against the host response. Due to the restricted movement of PMN, the bactericidal activity of PMN is only efficient against bacteria in the immediate vicinity, explaining the inefficient host defence.