Interaction of extracellular factors derived from Pseudomonas aeruginosa on keratinocytes

Pseudomonas aeruginosa is a Gram negative pathogenic bacterium that has the notable ability to inhabit a broad range of environments, including humans as an opportunistic pathogen. P aeruginosa secrets outer membrane vesicles (OMVs) which contain virulence factors such as: pro-elastase, hemolysin, phoshpolipase C, protease, alkaline phosphatase and B-lactamase that can damage the cells of host and also other bacteria. P aeruginosa commonly colonises wound beds and this can result in the development of a chronic wound which is an important cause of major pathology. Two strains of P aeruginosa PS3 (hospital strain), which was isolated from the dressing of an established chronic wound and a reference laboratory strain (L) that had no known pathogenic factors were cultured in different media including a simulated wound fluid. The secreted extracellular products were isolated and used to test their virulence on a cultured human keratinocyte cell line (HaCaT) and primary human keratinocytes (NHK). Vesicles derived from the outer membrane of the bacteria were isolated and the protein expression in these outer membrane vesicles (OMV) were compared between the two strains of P aeruginosa and with the outer membranes isolated from both strains of bacteria. The virulence of these vesicles and the outer membrane was tested on the keratinocytes. The initial part of the study demonstrated that HaCaT cells grown for either 4 or 10days expressed a range of TLRs required for the recognition and response to a wide range of bacterial antigens TLR1, TLR2, TLR4, and both aged cells. In contrast TLR5, TLR9 were found just in 10 days cells. The expression of a range of TLRs was investigated and the most expression at the mRNA level was found for TLR2 and TLR4 when the cells were grown for either 4 or 10 days. Both strains of P aeruginosa produced OMVs that appeared to contain a similar protein profile and this was similar to that of the isolated outer membrane. The yield OMVs from PS3 and the lab strain was generally similar but was higher when the bacteria were treated with gentamycin before isolation and also when the bacteria were grown in the presence of ethanol. Cultured keratinocyte cells secreted of IL-8 in response to exposure to cell free extracellular material from both strains P aeruginosa with little effect on the cell biomass. HaCaT cells produced a much higher concentration of IL-8 in response to OMVs than did NHK or an adapted more proliferative derivative of HaCaT (HaCaTa) with OMVs from the hospital strain stimulating slightly more IL-8 secretion compared to lab strain. NHK also produced significant amounts of IL-8 in response to challenge and with OM of PS3 or the lab strain. P. aeruginosa grown in SWF produced OMVs that stimulated a greater production of IL-8 in keratinocytes comparted to bacteria grown in normal bacterial broth (TSB). These data indicate that P aeruginosa which are a common bacterium isolated from the bed of chronic wounds secrete virulence factors that stimulate an inflammatory phenotype in cultured keratinocytes. This pathogenic response is largely driven by the secretion of vesicles derived from the outer membrane and the hospital strain has a slightly greater pathogenicity than the lab strain this has implications for the treatment of wounds.