the initial portion of this study has been focused on the characterization of the qteE mutant virulence-related phenotypes in test-tube cultures. Up coming, the result of equally qteE and rsaL mutations on P. aeruginosa pathogenesis has been investigated in a murine design of continual an infection. Benefits showed that a mutation in qteE causes a untimely QS response and hyperproduction of virulence aspects in P. aeruginosa cultures. Nonetheless, the anticipation of the QS response in the pre-quorum interval thanks to the qteE mutation does not impact P. aeruginosa pathogenicity, even though the put up-quorum dysregulation brought on by the rsaL mutation hampers the establishment of long-term lung infection. All round these results contribute to fill-in the present hole of knowledge about the relevance of QS modulation in P. aeruginosa pathogenesis, and promote a re-discussion of the all round position played by QS for the duration of the infection approach.
The impact of qteE mutation on P. aeruginosa 3OC12-HSLdependent response was established along development by comparing the ranges of this sign molecule and of picked QS-dependent virulence factors in wild kind and in qteE cultures carrying both the vacant vector pBBR1MCS-5 or its spinoff plasmid (named pQteE) expressing the qteE gene. As proven in Determine 2A, the qteE mutant produced detectable ranges of 3OC12-HSL previously than the wild variety pressure, reaching a 3OC12-HSL concentration about 6-fold higher at A600<1. Interestingly, 3OC12-HSL levels measured in the qteE and in the wild type strains plateaued at the same level in the postquorum phase of growth (A600<2). This trend of 3OC12-HSL production in the qteE mutant is also consistent with previous western hybridization experiments showing that the positive effect of the qteE mutation on LasR protein stability is restricted to the pre-quorum period [20]. Conversely, as previously shown [24], the rsaL mutant disclosed normal 3OC12-HSL production in the prequorum period, while this mutant produced higher 3OC12-HSL levels than the wild type strain after the QS threshold has been reached (A600.1.8 Fig. 2A). In agreement with the precocious synthesis of 3OC12-HSL, the qteE mutant also anticipated the production of elastase (Fig. 2B) and protease (Fig. 2C). Differently from 3OC12-HSL levels, it seems that the anticipated expression of proteases and elastase levels in the qteE mutant causes accumulation of these secreted factors also in the post-quorum period (compare panels A, B and C of Fig. 2). The homeostatic control of 3OC12-HSL levels in the post-quorum period is likely due to specific mechanisms that do not affect proteases and elastase production, including the transcriptional repression exerted by RsaL on lasI, and the activity of the acyl-HSL degrading enzymes produced by P. aeruginosa [15,23?5,32?4]. Also the biosynthesis of the cytotoxic secondary metabolite pyocyanin is activated by the LasR/3OC12-HSL complex, though it starts later during the growth with respect to proteases and elastase biosynthesis [35]. Interestingly, when the wild type and qteE mutant cultures reached an A600<3.5, the supernatants of the qteE mutant contained high pyocyanin levels, while this virulence factor was almost undetectable in the wild type strain (Fig. 3). The growth curve of the qteE mutant was similar to those of the rsaL mutant and wild type strains and was not affected by the presence of the pBBR1MCS-5 vector, ruling out the possibility that differences in the growth rates could account for the diverse phenotypes described above (Fig. S1 in File S1).
Effect of QS dysregulation caused by qteE mutation on P. aeruginosa virulence-related phenotypes. Levels of (A) 3OC12-HSL, (B) elastase, (C) proteases produced along growth by P. aeruginosa wild type (black lines), qteE (red lines) and rsaL strains (green line) carrying the pBBR1MCS-5 empty vector, or by the qteE strain carrying the pQteE plasmid (pBBR1MCS-5-derived) for the expression of qteE (dashed red line). Values are the means (6 standard deviations) of at least three independent experiments.
