Green Fluorescent Protein-Expressing<i>Escherichia coli</i>as a Selective Probe for HOCl Generation within Neutrophils

Palazzolo, Amy M; Suquet, Christine; Konkel, Michael E.; Hurst, James K.

doi:10.1021/bi047342s

Cited by 44 publications

(51 citation statements)

References 63 publications

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“…2, A and B). Moreover, these concentrations have been estimated previously to exist within neutrophil phagosomes (31,(33)(34)(35)(36)(37); albeit, recent studies indicate the steady-state concentration of H 2 O 2 is much lower (38). As with the concentration-dependent killing of S. aureus by either H 2 O 2 or HOCl, there was limited time-dependent effect on viability (i.e., Ͻ100% survival) of MW2 following exposure to these microbicides (Fig.…”

Section: S Aureus Is Resistant To Major Neutrophil Microbicidesmentioning

confidence: 79%

Neutrophil Microbicides Induce a Pathogen Survival Response in Community-Associated Methicillin-Resistant Staphylococcus aureus

Palazzolo-Ballance¹,

Reniere

Braughton³

et al. 2008

The Journal of Immunology

130

120

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In recent years, there has been a dramatic increase in the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections. MW2 (pulsed-field type USA400), the prototype CA-MRSA strain, is highly virulent and has enhanced ability to evade killing by neutrophils. Although progress has been made, the molecular basis for enhanced virulence of CA-MRSA remains incompletely defined. To that end, we studied resistance of MW2 to key microbicides of human neutrophils. Hydrogen peroxide (H2O2), hypochlorous acid, and azurophilic granule proteins had significant bacteriostatic but limited staphylocidal activity toward MW2 under the conditions tested. An MW2-specific microarray revealed common changes in S. aureus gene expression following exposure to each microbicide, such as up-regulation of transcripts involved in gene regulation (e.g., saeRS and kdpDE) and stress response. Azurophilic granule proteins elicited the greatest number of changes in MW2 transcripts, including up-regulation of mRNAs encoding multiple toxins and hemolysins (e.g., hlgA, hlgB, hlgC, hla, lukS-PV, lukF-PV, sec4, and set17–26). Notably, H2O2 triggered up-regulation of transcripts related to heme/iron uptake (e.g., isdA, isdB, and isdCDEFsrtBisdG), and an isogenic isdAB-negative strain of MW2 had increased susceptibility to H2O2 (p < 0.001) and human neutrophils (p < 0.05) compared with the wild-type parental strain. These findings reveal a S. aureus survival response wherein Iron-regulated surface determinant (Isd) proteins are important for resistance to innate host defense. Collectively, the data provide an enhanced view of the mechanisms used by S. aureus to circumvent destruction by the innate immune system.

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Section: S Aureus Is Resistant To Major Neutrophil Microbicidesmentioning

confidence: 79%

Neutrophil Microbicides Induce a Pathogen Survival Response in Community-Associated Methicillin-Resistant Staphylococcus aureus

Palazzolo-Ballance¹,

Reniere

Braughton³

et al. 2008

The Journal of Immunology

130

120

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“…Furthermore, as noted above, Mayer and coworkers have reported widespread tyrosyl nitration within LPS/IFγ-activated RAW cells within roughly the same time scale (26); although in this case the target sites were not identified, they must have involved other intracellular proteins. These data suggest that, like all other heme peroxidases that have been investigated (38,70), COX-2 can oxidize available NO 2 -at its peroxidase site (71) via peroxide-generated compounds I and II to NO 2 · , which is then diffusible from the active site. We recently found that NO 2 · is remarkably toxic to Escherichia coli, with an LD 50 comparable to that of the much more strongly oxidizing and potently microbicidal CO 3 ·-radical anion (28), and Klebanoff has demonstrated that E. coli are also effectively killed when exposed to a cell-free MPO-H 2 O 2 -NO 2 -system (70).…”

Section: Physiological Implicationsmentioning

confidence: 79%

Pathways for Intracellular Generation of Oxidants and Tyrosine Nitration by a Macrophage Cell Line

2007

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Two transformed murine macrophage cell lines (RAW 264.7 ATCC TIB-71 and CRL-2278) were examined for oxidant production at various times following activation by using a set of fluorescence and ESR-active probes. Stimulation with a soluble agonist or activation with bacterial lipopolysaccharide plus γ-interferon caused only very small initial increases in O 2 consumption above basal rates; however, at 2-4 h post-activation, respiration increased to 2-3 fold and remained at these elevated levels over the subsequent lifetime of the cell (20-30 h). Oxidation reactions were confined primarily within the cell, as was demonstrated by using phagocytosable dichlorodihydrofluorescein-conjugated latex beads and cyclic hydroxylamines with differing membrane permeabilities. From the intrinsic reactivities of these probes and the time course of their oxidations, one infers induction of apparent peroxidase activity beginning at ∼2 h post-activation, coinciding with the increase in overall respiratory rate; this acquired capability was accompanied by accumulation of a stable horseradish peroxidase-reactive oxidant, presumably H 2 O 2 , in the extracellular medium,. Nitrite ion rapidly accumulated in the extracellular medium over a period of 5-8 h post-activation in both cell lines, indicating the presence of active nitric oxide synthase (iNOS) during that period. Prostaglandin endoperoxide H synthase (COX-2) activity was detected at 15-20 h post-activation by use of sensitive peroxide assay in conjunction with a COX-2 specific inhibitor (DuP-697). Superoxide formation was detected by reaction with hydroethidine within the first hour following activation, but not thereafter. Consistent with the absence of significant respiratory stimulation, the amount of O 2 ·-formed was very small; comparative reactions of cyclic hydroxylamine probes indicated that virtually none of the O 2 ·-was discharged into the external medium. Myeloperoxidase (MPO) activity was probed at various times post-activation by using fluorescein-conjugated polyacrylamide beads, which efficiently trap MPO-generated HOCl in neutrophils to give stable chlorofluorescein products. However, chlorination of the dye was not detected under any conditions in RAW cells, virtually precluding MPO involvement in their intracellular reactions. This same probe was used to determine changes in intraphagosomal pH, which increased slowly from ∼6.5 to ∼8.2 over a 20 h post-phagocytosis period. The cumulative data suggest activation is followed by sequential induction of an endogenous peroxidase, iNOS, and COX-2, with NADPH oxidase-derived O 2 ·-playing a minimal role in direct generation of intracellular oxidants. To account for reported observations of intracellular tyrosine nitration late in the life cycles of macrophages, we propose a novel mechanism wherein iNOS-generated NO 2 -is used by COX-2 to produce NO 2 · as a terminal microbicidal oxidant and nitrating agent.The existence of motile phagocytic cells involved with host defense in higher organisms has been known sin...

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“…PMN phagocytosed bacteria are usually killed rapidly (14,18), and we therefore set out to investigate the fate of phagocytosed Ag43-expressing bacteria. Accordingly, the survival rate of such bacteria was monitored for 120 min (Fig.…”

Section: Resultsmentioning

confidence: 99%

Biological Trojan Horse: Antigen 43 Provides Specific Bacterial Uptake and Survival in Human Neutrophils

et al. 2007

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Escherichia coli is a versatile pathogen causing millions of infections in humans every year. This bacterium can form multicellular aggregates when it expresses a self-associating protein, antigen 43 (Ag43), on its surface. We have discovered that Ag43-expressing E. coli cells are efficiently taken up by human defense cells, polymorphonuclear neutrophils (PMNs), in an opsonin-independent manner. Surprisingly, the phagocytosed bacteria were not immediately killed but resided as tight aggregates within the PMNs. Our observations indicate that Ag43-mediated uptake and survival in PMNs constitute a mechanism to subvert one of the primary defense mechanisms of the human body.

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Green Fluorescent Protein-ExpressingEscherichia colias a Selective Probe for HOCl Generation within Neutrophils

Cited by 44 publications

References 63 publications

Neutrophil Microbicides Induce a Pathogen Survival Response in Community-Associated Methicillin-Resistant Staphylococcus aureus

Neutrophil Microbicides Induce a Pathogen Survival Response in Community-Associated Methicillin-Resistant Staphylococcus aureus

Pathways for Intracellular Generation of Oxidants and Tyrosine Nitration by a Macrophage Cell Line

Biological Trojan Horse: Antigen 43 Provides Specific Bacterial Uptake and Survival in Human Neutrophils

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