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

Abstract

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 TM , 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 TM , 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 21 is not required for cytoplasmic Ca 21 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. ' International Society for Advancement of CytometryKey terms calcium; pH; reactive oxygen species PHAGOCYTIC cells, such as polymorphonuclear (PMN) neutrophils and tissue macrophages (MP), are essential components of the human innate defense against infection. Phagocyte surface receptors recognize invading bacteria by a variety of mechanisms, including complement-dependent opsonization, antibody/antigen binding via Fc receptors, or pattern recognition (e.g., mannose-, scavenger-, or Toll-) receptor liganding. They then signal, via changes in intracellular cation concentrations, in transmembrane potentials, and in secondary signaling (e.g., via activation of specific phospholipases) so as to initiate a series of processes that result in phagocytosis with subsequent intraphagosomal killing and clearance of the organism [for some very recent reviews see (1-7)].Many of these signaling events occur early, within seconds of ligand-receptor binding, and have been shown to vary with the specific receptors involved (8)(9)(10)(11)(12)(13)(14). Yet, the changes initiated by these signals to generate effector functions of the cell do not become measurable by conventional methods until at least minutes later. Furthermore, responses to such stimulation...

Phagocytosis of neonatal pathogens by peripheral blood neutrophils and monocytes from newborn preterm and term infants

“…These inconsistent findings are likely to reflect methodological differences, as sample type (e.g., purified cells or whole blood), preparations and species of bacteria, together with the use of humoral factors (e.g., complement) vary considerably between studies (11). Although previous studies have utilized pHrodo as a tool for assessing phagocytosis, none have extended its use to simultaneous analysis of multiple cell types, or to whole blood assays (18,21). We found that preterm infants possess a decreased proportion of whole-blood phagocytes capable of phagocytosing the common neonatal pathogens SE, EC and, to a lesser extent, SA compared with term infants.…”

“…One possible caveat to these findings is the necessary use of heat-killed bacterial preparations, and commercially prepared SA and EC stocks that are non-neonatal sepsis isolates, which may influence interactions (33). Future studies could examine the real-time phagocytosis of live, neonatal bacterial isolates by infant monocytes and neutrophils (18). However, this would first require modification and optimization of the methodologies used, in order to ensure they are compatible with the very small postnatal blood volumes available from extremely preterm infants.…”

“…The formation of the phagosome around an ingested entity during phagocytosis relies on low pH for bactericidal functions and antigen processing and presentation (1,18). pHrodo is a highly pH-sensitive succinimidyl ester dye that fluoresces dramatically when exposed to low pH (<4.0).…”

“…pHrodo is a highly pH-sensitive succinimidyl ester dye that fluoresces dramatically when exposed to low pH (<4.0). Phagocytosis is therefore detectable and quantifiable when the phagosome forms, and acidifies, around ingested pHrodo-labeled bacteria, minimizing high background fluorescence of cell-bound, but uningested, bacteria (18,19).…”

Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification

“…Analysis of the pH of S. Typhimurium SCVs was performed according to a previous report (Wong et al, 2011). Bacteria were either dually labelled with a pH-sensitive probe (pHrodo, which emits red fluorescence in an acidic environment; Invitrogen) and a pH-insensitive probe Alexa Fluor 488 (green) or singly with pH-sensitive FITC as described previously (Bernardo et al, 2010). The calculated fluorescence ratios of pH-sensitive and pH-insensitive probes (in the dual-labelling process) and mean fluorescence intensity (in the single-labelling process) were used to determine phagosomal pH according to a calibration curve.…”