S. aureus has been shown to be a facultative intracellular pathogen that can invade human non-professional phagocytes. Many aspects of the processes following invasion are only partially understood. Especially, the fate and maturation of the S. aureus-containing phagosome remain controversial.
We used a novel flow cytometric assay based on the pH-dependent quenching of FITC fluorescence to follow the phagosomal acidification over time in three different human cell lines (HEK 239, Ea.Hy926, HeLa), as well as lysotracker colocalization studies.
Comparison of fluorescence intensity in the absence and presence of monensin, a Na⁺/H⁺ ionophore, allowed indirect pH measurement of S. aureus-containing phagosomes. In a kinetic analysis, acidification could be observed to reach a maximum at 2h, subsequently remaining constant over a time period of up to 24h. By contrast, neutralizing agents including NH₄Cl, chloroquine and the vesicular H⁺-ATPase inhibitor bafilomycin A1 showed dose-response curves. We analyzed the phagosomal pH over time (at 2 and 6h) in a collection of S. aureus laboratory strains and clinical isolates (MRSA and MSSA). In all but one strains examined, we observed a maintained low phagosomal pH, indicating the functional integrity of the membrane. In no case the pH was completely neutralized. In addition, lysotracker colocalization studies corroborated an acidification of the phagosomal compartment.
Taken together, these data suggest, that the phagosomal compartment stays functionally intact for up to 24h, which clearly argues against a destruction of the phagosomal membrane. This is in contrast to previously published (and own unpublished) data, suggesting morphological alterations, compatible with a phagosomal escape as early as 3 to 6h post invasion.