In the supernatant of the mutant, high molecular mass bands matching different Palbociclib price forms of the major S. aureus autolysin Atl [35], were expressed similarly (>130 kDa, pro-Atl) or even stronger (~84 kDa, PP-AM) compared to the wild type and the complemented mutant (Figure 5B). Interestingly, the >130 kDa band migrated at a slightly

higher position in the mutant, corresponding to the height of the pro-Atl band in the cell wall fractions, where the mutant showed overall stronger hydrolytic bands than wild type or complemented mutant. Deletion of secDF leads to altered expression of virulence factors We qualitatively assessed the amount of various Sec-dependent S. aureus virulence factors, such as coagulase, hemolysin and protease activities, as well as of the immunomodulatory protein SpA to determine whether they were affected in the secDF mutant as well. Supernatant from Newman and the complemented secDF mutant coagulated rabbit plasma after 30 min, whereas

the secDF mutant required 90 min, suggesting production of slightly reduced coagulase levels. Extracellular proteolytic activity seemed to be absent in the secDF mutant, even after five days of incubation, PF-02341066 supplier whereas cultures from Newman and the complemented mutant showed distinct proteolytic halos on skim milk agar after 72 h (Figure 6A). Hemolysis activity was tested by a similar agar diffusion assay as used for protease activity determination. Overnight Etomoxir cultures, or sterile-filtrated culture supernatants, were dispensed into holes on sheep blood agar. Newman and the complemented secDF mutant showed the same extent of hemolysis. In the secDF background hemolysis was reduced when bacteria grew on the rim of agar

holes (Figure 6B), but was increased when the hemolytic activity of sterile supernatant from liquid cultures was tested (Figure 6C and 6D). Sessile or planktonic growth affects regulatory mechanisms, which can alter the expression of virulence factors such as Hla [36, 37]. Here we found that the deletion of secDF DNA ligase had divergent effects on hemolysin expression depending on the growth conditions, most likely by affecting regulatory processes. Figure 6 Proteolysis and hemolysis of sessile and planktonic bacteria. Proteolytic and hemolytic activity was determined qualitatively by agar diffusion assay on skim milk, respectively sheep blood agar. Hemolytic activity was measured in diluted sheep blood. (A) Skim milk agar and (B) sheep blood agar with sessile bacteria. (C) Sheep blood agar with sterile-filtered supernatants of stationary phase planktonic bacteria. (D) Release of hemoglobin by stationary phase supernatants of planktonic bacteria. Representative data of three independent experiments are shown with standard deviations of technical triplicates. SpA is one of the proteins predicted to be attached to the cell wall by sortase following export [38].