Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis

Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis ( Mtb ), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within the phagosome to later cause disease. To study such evasion, we developed protocols which permit simultaneous kinetic measurement of early cytoplasmic signaling and of phagosomal pH (pH p ) and oxidative burst, on a cell‐by‐cell basis, of polymorphonuclear (PMN) leukocytes exposed to fluorescently labeled, nonpathogenic Staphylococcus epidermidis ( Se ). The availability of a new, highly sensitive pH probe, pHrodo™, permits observation of increasing pH p . Simultaneous labeling of the organism, applicable to any phagocyte target, with a probe insensitive to pH and oxidative species, such as AlexaFluor350™, permits distinction between binding and functional responses to it by ratioing fluorescences. Addition of an extracellular‐specific quencher (Trypan blue) permits distinction between bound and phagosome‐enclosed targets, so that conditions within the closed phagosome can be studied. We found that opsonization is required for functional activation of PMN by Se , that the organism causes early alkalinization of the phagosome (in contrast to Cf which hyperacidifies it), and that extracellular Ca 2+ is not required for cytoplasmic Ca 2+ signaling but contributes markedly to binding of Se to PMN and to ensuant bactericidal functions. These findings lead to a new approach to the study of select organisms, like Cf and Mtb , which evade killing by manipulating the phagosomal environment. © 2009 International Society for Advancement of Cytometry