No abstract

Phagocytes such as neutrophils play a key role in the body's innate immune response to infection. These cells travel throughout the body in search of pathogens and are rapidly mobilized to sites of inflammation where they phagocytose these pathogens and subsequently release a variety of toxic oxygen radical species and proteolytic enzymes to directly destroy the engulfed particle. The generation of microbicidal oxidants by neutrophils results from the action of a multi-protein enzymatic complex known as the NADPH oxidase. Altogether, there are currently seven proteins reported to be associated with the NADPH oxidase assembly. In resting neutrophils, these NADPH oxidase protein components are segregated into cytoplasmic and plasma membrane compartments. However, during assembly and activation of the NADPH oxidase, the cytosolic protein components translocate to the plasma membrane or phagosomal membrane where they assemble around a central membrane-bound protein known as flavocytochrome b. This assembly process is highly regulated and involves multiple binding interactions between the individual NADPH oxidase proteins, resulting in an active oxidase complex. Over the past few years, a number of these sites of binding interaction between the oxidase proteins have been identified, leading to a clearer understanding of the intermolecular interactions occurring among protein components during the assembly process. In addition, this information has contributed to our understanding of the roles played by each protein during the activation and assembly process. In this review, we describe the key features of each NADPH oxidase protein and then summarize our current understanding of the specific molecular interactions occurring between these proteins, focusing on the role these protein:protein binding interactions play in the NADPH oxidase assembly process.

Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.

Polymorphonuclear leukocytes (PMNs or neutrophils) are essential to human innate host defense. However, some bacterial pathogens circumvent destruction by PMNs and thereby cause disease. Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, survives within PMNs in part by altering normal host cell processes, such as production of reactive oxygen species (ROS) and apoptosis. To investigate the molecular basis of A. phagocytophilum survival within neutrophils, we used Affymetrix microarrays to measure global changes in human PMN gene expression following infection with A. phagocytophilum. Notably, A. phagocytophilum uptake induced fewer perturbations in host cell gene regulation compared with phagocytosis of Staphylococcus aureus. Although ingestion of A. phagocytophilum did not elicit significant PMN ROS, proinflammatory genes were gradually up-regulated, indicating delayed PMN activation rather than loss of proinflammatory capacity normally observed during phagocytosis-induced apoptosis. Importantly, ingestion of A. phagocytophilum failed to trigger the neutrophil apoptosis differentiation program that typically follows phagocytosis and ROS production. Heat-killed A. phagocytophilum caused some similar initial alterations in neutrophil gene expression and function, which included delaying normal PMN apoptosis and blocking Fas-induced programmed cell death. However, at 24 h, down-regulation of PMN gene transcription may be more reliant on active infection. Taken together, these findings suggest two separate antiapoptotic processes may work concomitantly to promote bacterial survival: 1) uptake of A. phagocytophilum fails to trigger the apoptosis differentiation program usually induced by bacteria, and 2) a protein or molecule on the pathogen surface can mediate an early delay in spontaneous neutrophil apoptosis.

Human polymorphonuclear leukocytes (PMNs or neutrophils) kill invading microorganisms with reactive oxygen species (ROS) and cytotoxic granule components. PMNs from individuals with X-linked chronic granulomatous disease (XCGD) do not produce ROS, thereby rendering these individuals more susceptible to infection. In addition, XCGD patients develop tissue granulomas that obstruct vital organs, the mechanism(s) for which are unknown. To gain insight into the molecular processes that contribute to the pathophysiology of XCGD, including formation of granulomas, we compared global gene expression in PMNs from XCGD patients and healthy control individuals. Genes encoding mediators of inflammation and host defense, including CD11c, CD14, CD54, FcγR1, FcαR, CD120b, TLR5, IL-4R, CCR1, p47phox, p40phox, IL-8, CXCL1, Nramp1, and calgranulins A and B, were up-regulated constitutively in unstimulated XCGD patient PMNs. By comparing transcript levels in normal and XCGD PMNs after phagocytosis, we discovered 206 genes whose expression changed in the presence and the absence of ROS, respectively. Notably, altered Bcl2-associated X protein synthesis accompanied defective neutrophil apoptosis in XCGD patients. We hypothesize that granuloma formation in XCGD patients reflects both increased proinflammatory activity and defective PMN apoptosis, and we conclude that ROS contribute directly or indirectly to the resolution of the inflammatory response by influencing PMN gene transcription.

Polymorphonuclear leukocytes (PMNs) are essential to innate immunity in humans and contribute significantly to inflammation. Although progress has been made, the molecular basis for termination of inflammation in humans is incompletely characterized. We used human oligonucleotide microarrays to identify genes encoding inflammatory mediators that were differentially regulated during the induction of apoptosis. One hundred thirty-three of 212 differentially expressed genes encoding proinflammatory factors, signal transduction mediators, adhesion molecules, and other proteins that facilitate the inflammatory response were down-regulated during the induction of apoptosis following PMN phagocytosis. Among these, 42 genes encoded proteins critical to the inflammatory response, including receptors for IL-8β, IL-10α, IL-13α1, IL-15α, IL-17, IL-18, C1q, low-density lipoprotein, IgG Fc (CD32), and formyl peptide, Toll-like receptor 6, platelet/endothelial cell adhesion molecule-1 (CD31), P-selectin (CD62), IL-1α, IL-16, and granulocyte chemoattractant protein-2 were down-regulated. Many of these genes were similarly down-regulated during Fas-mediated or camptothecin-induced apoptosis. We used flow cytometry to confirm that IL-8Rβ (CXCR2) and IL-1α were significantly down-regulated during PMN apoptosis. We also discovered that 23 genes encoding phosphoinositide and calcium-mediated signal transduction components, which comprise complex pathways essential to the inflammatory response of host cells, were differentially regulated during PMN apoptosis. Importantly, our data demonstrate that PMNs down-regulate proinflammatory capacity at the level of gene expression during induction of apoptosis. These findings provide new insight into the molecular events that resolve inflammation following PMN activation in humans.

Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity ( ‫؍‬ 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.The ability of Staphylococcus aureus to evade the host immune response and cause disease is due to an extensive repertoire of known and putative virulence factors, including four hemolysins, two lipases, several proteases, exotoxins, and enterotoxins. The production of many virulence factors is regulated by the accessory gene regulatory (agr) operon (22,27) and several other global regulatory loci. The agr locus consists of two divergently transcribed mRNAs designated RNA II and RNA III (19). RNA II encodes a two-component regulatory system (AgrA and AgrC) that senses the level of cyclic thiolactone peptides generated from agrB and agrD, also encoded by RNA II (15). RNA III is an RNA effector molecule that reciprocally regulates the transcription of cell-associated adherence factors and secreted proteins (25). RNA III also regulates the translation of alpha-toxin mRNA (25). Transcription of the agr operon is autoregulated by agrA in a cell densitydependent manner and by at least two other global regulatory proteins: the staphylococcal accessory regulator (SarA) (6) and ArlR, the regulatory moiety of the ArlS-ArlR two-component regulatory system (10). Recently, we determined that aconitase affects the synthesis of several S. aureus virulence factors and the expression of the global gene regulators RNA III and sarA (G. A. Somerville, unpublished data). Aconitase (citrate [isocitrate] hydrolyase, EC 4.2.1.3) is a citric acid cycle enzyme ...

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 &lt; 0.001) and human neutrophils (p &lt; 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.