Staphylococcal Alpha-Toxin Is Not Sufficient To Mediate Escape from Phagolysosomes in Upper-Airway Epithelial Cells

Abstract

Intracellular Staphylococcus aureus has been implicated in the establishment of chronic infections. It is therefore imperative to understand by what means S. aureus is able to survive within cells. Here we use two expression systems with a fluorescent readout to assay alpha-toxin expression and function within phagolysosomes of infected upper-airway epithelial cells: avirulent Staphylococcus carnosus TM300 and phenotypically alpha-toxin-negative S. aureus laboratory strains. Data from CFU recovery assays suggest that the presence of alpha-toxin is not beneficial for the intracellular survival of recombinant Staphylococcus strains. This finding was corroborated by immunofluorescence studies: whereas S. carnosus and S. aureus are able to deliver S. aureus alpha-toxin to lumina of host cell phagolysosomes, the membrane integrity of these organelles was not affected. Alpha-toxin-expressing strains were detected exclusively within lysosome-associated membrane protein 1 (LAMP1)-yellow fluorescent protein (YFP)-positive vesicles. Measurements of intraphagosomal pH illustrated that all infected phagolysosomes acidified regardless of alpha-toxin expression. In contrast, S. aureus expressing Listeria monocytogenes listeriolysin O leads to the breakdown of the phagolysosomal membrane, as indicated by staphylococci that are not associated with LAMP1-YFP-decorated vesicles and that do not reside within an acidic cellular environment. Thus, our results suggest that staphylococcal alpha-toxin is not sufficient to mediate phagolysosomal escape in upper-airway epithelial cells.

Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin againstStaphylococcus aureus

“…This property further increases delafloxacin's potency in acidic environments (35,43), as also observed for N-sulfanilylpiperazinyl fluoroquinolones (1,3) and finafloxacin (21). This could be an advantage with respect to S. aureus infections, since this bacterium, which shows a high tolerance to low pH, survives and multiplies in mild acidic environments (47), such as the skin, the vagina, or the urinary tract, and within the phagolysosomes of infected cells (6), where the pH is about 5 to 5.5 (19). Thus, delafloxacin contrasts with what is observed for many of the current antistaphylococcal antibiotics for which activity is reduced when the pH is lowered, as seen for other fluoroquinolones (e.g., norfloxa-cin, ciprofloxacin, or moxifloxacin [6,48]), macrolides (26), clindamycin (37), and gentamicin (7).…”

Phagolysosomal Integrity Is Generally Maintained afterStaphylococcus aureusInvasion of Nonprofessional Phagocytes but Is Modulated by Strain 6850

“…Recently, a study reported a crucial role for ␣-toxin in phagosomal escape (20), using LAMP decoration. Staphylococcal ␣-toxin had been implicated in phagolysosomal escape in cystic fibrosis airway epithelial cells (20) but not in cells complemented with a functional cystic fibrosis transmembrane conductance regulator (CFTR) (12,20). Thus, due to the absence of any correlation between the hemolytic activity and the ability to modulate the phagosomal pH, our previous results (12) suggest the existence of an effector other than ␣-toxin, involved in the modulation of phagosomal pH by strain 6850 (Fig.…”

“…6A). We examined whether the bacteria were surrounded by a phagolysosomal membrane by infecting HeLa cells that expressed the lysosomal membrane marker LAMP-1 fused to YFP (12). Of all investigated strains, only strain 6850 resulted in a few bacteria that were devoid of a LAMP1-YFP-labeled membrane envelope (Fig.…”

Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus

“…1a). According to previous experiences (Christner et al, 2010;Giese et al, 2009), these vectors can be assumed to also function efficiently in other staphylococci.…”