aeruginosa PAO1 [22]. To further investigate the involvement of TypA in the pathogenesis of P. aeruginosa, we constructed a site-directed typA knock-out mutant in P. aeruginosa strain Erismodegib nmr PA14. Strain PA14 is capable of infecting a wide range of organisms including

the amoeba D. discoideum[23, 24] and the nematode C. elegans[4] and was therefore more suitable for virulence analysis using in vivo model systems in comparison to strain PAO1. Detailed analyses of virulence attenuation of the PA14 typA mutant using the unicellular eukaryotic model organism D. discoideum revealed a consistent, statistically significant (P < 0.001 by Mann Whitney test) 2-fold reduction in the numbers of amoebae required to form a plaque when compared to wild type strain PA14 (Figure 1).

The virulence phenotype could be completely restored CP-690550 price to wild type level by heterologous expression of the cloned typA gene in strain PA14 typA::ptypA + . In comparison, a similar 2-fold reduction in numbers of amoebae was determined when analyzing PA14 transposon mutant ID29579 obtained from the Harvard PA14 mutant library [25] with a defect in the pscC gene, which is an essential part of the Type III secretion system machinery [26], as a control (Figure 1). To exclude the fact that a simple growth deficiency of the typA mutant is responsible for the attenuated virulence phenotype of PA14 typA, we performed growth analyses at 23°C and 37°C in M9 minimal medium using a Tecan plate reader under shaking conditions. At both temperatures no significant growth defect was observed (data not shown). Figure 1 D. discoideum plate killing assay. Each point represents the number of amoebae required to form a plaque on the bacterial lawn of P. aeruginosa PA14 strains after 5 days of incubation.

The typA and pscC mutants had a major defect in this virulence model of infection, which was statistically significant as measured with the Mann Whitney test (*** p < 0.001, n = 9). Since phagocytosis of pathogens by macrophages is a crucial factor in the human immune defense system, we quantitatively analyzed in vitro uptake of Reverse transcriptase PA14 WT and respective mutant strains using human macrophages in a gentamicin protection assay. We determined a more than 2-fold increase in internalization of the typA and the pscC mutant strain in comparison to cells of PA14 WT and complemented strain PA14 typA::ptypA + (Figure 2). This result was in accord with the virulence defect observed in the amoeba model of infection, which is similarly based on phagocytic killing of bacterial cells. Figure 2 Uptake of P. aeruginosa by human macrophages. Strains were incubated with 1.5 × 105 cells/ml macrophages for 1 h at an MOI of 10.