FIGURE 8.

High PAE secretion and cortisol concentration-dependent growth inhibition of virulent P. aeruginosa may explain the relatively weak RCL potency of PAE. A, when cultured overnight, virulent P. aeruginosa (PASS1) isolated from the sputum of a cystic fibrosis patient showed, relative to the equivalent number of (or equivolume media from) the laboratory PAO1 wound strain, the following: a higher PAE secretion as evaluated by gel band intensities of filtered (F) and crude (C) culture media (i); significantly higher intracellular PAE levels as evaluated by intensity-based LC-MS/MS analysis of bacteria cell lysates (ii); and a higher capacity for generating the CBG-Ct fragments, indicating enhanced RCL cleavage (iii). ***, p < 0.001 (mean ± S.D. (error bars), n = 3). B, the PASS1 growth profile was monitored over time (0–20 h) without (black) and with various concentrations of cortisol (0.01–10 μm, various shadings of brown) covering the physiological cortisol range in humans. No significant differences were observed for the 0.01 μm cortisol relative to the untreated control (p ≥ 0.05 for all time points), whereas a minor concentration-dependent bacteriostatic growth inhibition was observed for the 0.1–10 μm cortisol relative to the 0.01 μm cortisol concentration (p < 0.05 for time points marked with an asterisk in the expanded inset). All data points are presented as mean ± S.E. (error bars) (n = 3). C, proposed simplistic model of the relationship between the human host inflammation and bacterial infection pathways in the context of NE- and PAE-induced cortisol release upon RCL cleavage. The potent NE acts preferentially on non-glycosylated Asn³⁴⁷ variants or CBG glycoforms carrying Asn³⁴⁷ glycans that are sialylated, non-core-fucosylated, and biantennary branched on the second-to-minute time scale, yielding a substantial release of cortisol from hepatocyte- and potentially extrahepatocyte-derived CBG present at the site of inflammation (24). In contrast, the less potent PAE acts exclusively on non-glycosylated Asn³⁴⁷ CBG variants during the longer (minutes-to-hours) incubation period and benefits significantly from the sialylation of non-Asn³⁴⁷ glycans. This rather inefficient proteolysis may generate a slow release of cortisol of only a subset of CBG-cortisol complexes, which may be below the bacteriostatic concentration threshold but perhaps still sufficient to reduce the host immune response by the anti-inflammatory effects of cortisol, thereby possibly creating a favorable infection environment of P. aeruginosa. The comparative faster and more substantial release of cortisol by NE, on the contrary, would, in principal, create a more rapid resolution of the inflammation and possibly generate a significant bacteriostatic effect and a more hostile environment for bacterial infection. See Fig. 1 and supplemental Table S1 for the nomenclature used for the Asn³⁴⁷ glycoforms.