Oxidative bursts of single mitochondria mediate retrograde signaling toward the ER.

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP3Rs), we show that Ca2+ channels directly sense oxidative bursts and respond with Ca2+ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca2+ transport via elimination of IP3Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca2+ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.

Defective neutrophil development and specific granule deficiency caused by a homozygous splice-site mutation in SMARCD2.

BACKGROUND: SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) has recently been shown to have a critical role in granulopoiesis in humans, mice, and zebrafish. Our patient presented with delayed cord separation, failure to thrive, and sepsis. Retrospective whole-exome sequencing confirmed a homozygous splice-site mutation in SMARCD2. OBJECTIVE: We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells. METHODS: Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34+ cells were then isolated, phenotyped, and assessed functionally. RESULTS: Circulating neutrophils appeared phenotypically immature, lacking multilobed nuclei, and neutrophil granules lacked lactoferrin but showed normal levels of myeloperoxidase. Neutrophil oxidative burst was preserved in response to phorbol 12-myristate 13-acetate. Patient bone marrow-derived neutrophils and white blood cells showed a severely impaired chemotactic response. Furthermore, white blood cells showed defective in vitro killing of Staphylococcus aureus, consistent with a specific granule deficiency. Finally, patient bone marrow-derived CD34+ cells showed markedly impaired in vitro expansion and differentiation toward the neutrophil lineage. Before her molecular diagnosis, our patient underwent hematopoietic stem cell transplantation and is well 8 years later. CONCLUSIONS: This report highlights an important role for SMARCD2 in human myelopoiesis and the curative effect of hematopoietic stem cell transplantation for the hematopoietic features of SMARCD2 deficiency.

"Oral Manifestations of Patients with Inherited Defect in Phagocyte Number or Function" a systematic review.

INTRODUCTION: Inherited phagocyte defects are one of the subgroups of primary immunodeficiency diseases (PIDs) with various clinical manifestations. As oral manifestations are common at the early ages, oral practitioners can have a special role in the early diagnosis. MATERIALS AND METHODS: A comprehensive search was conducted in this systematic review study and data of included studies were categorized into four subgroups of phagocyte defects, including congenital neutropenia, defects of motility, defects of respiratory burst, and other non-lymphoid defects. RESULTS: Among all phagocyte defects, 12 disorders had reported data for oral manifestations in published articles. A total of 987 cases were included in this study. Periodontitis is one of the most common oral manifestations. CONCLUSION: There is a need to organize better collaboration between medical doctors and dentists to diagnose and treat patients with phagocyte defects. Regular dental visits and professional oral health care are recommended from the time of the first primary teeth eruption in newborns.

SlREM1 Triggers Cell Death by Activating an Oxidative Burst and Other Regulators.

Programmed cell death (PCD), a highly regulated feature of the plant immune response, involves multiple molecular players. Remorins (REMs) are plant-specific proteins with varied biological functions, but their function in PCD and plant defense remains largely unknown. Here, we report a role for remorin in disease resistance, immune response, and PCD regulation. Overexpression of tomato (Solanum lycopersicum) REMORIN1 (SlREM1) increased susceptibility of tomato to the necrotrophic fungus Botrytis cinerea and heterologous expression of this gene triggered cell death in Nicotiana benthamiana leaves. Further investigation indicated that amino acids 173 to 187 and phosphorylation of SlREM1 played key roles in SlREM1-triggered cell death. Intriguingly, multiple tomato REMs induced cell death in N benthamiana leaves. Yeast two-hybrid, split luciferase complementation, and coimmunoprecipitation assays all demonstrated that remorin proteins could form homo- and heterocomplexes. Using isobaric tags for relative and absolute quantitative proteomics, we identified that some proteins related to cell death regulation, as well as N benthamiana RESPIRATORY BURST OXIDASE HOMOLOG B (which is essential for reactive oxygen species production), were notably upregulated in SlREM1-expressing leaves. Heterologous expression of SlREM1 increased reactive oxygen species accumulation and triggered other cell death regulators. Our findings indicate that SlREM1 is a positive regulator of plant cell death and provide clues for understanding the PCD molecular regulatory network in plants.

Analysis of the Oxidative Burst and Its Relevant Signaling Pathways in Leptosphaeria maculans-Brassica napus Pathosystem.

An oxidative burst is an early response of plants to various biotic/abiotic stresses. In plant-microbe interactions, the plant body can induce oxidative burst to activate various defense mechanisms to combat phytopathogens. A localized oxidative burst is also one of the typical behaviors during hypersensitive response (HR) caused by gene-for-gene interaction. In this study, the occurrence of oxidative burst and its signaling pathways was studied from different levels of disease severity (i.e., susceptible, intermediate, and resistant) in the B. napus-L. maculans pathosystem. Canola cotyledons with distinct levels of resistance exhibited differential regulation of the genes involved in reactive oxygen species (ROS) accumulation and responses. Histochemical assays were carried out to understand the patterns of H2O2 accumulation and cell death. Intermediate and resistant genotypes exhibited earlier accumulation of H2O2 and emergence of cell death around the inoculation origins. The observations also suggested that the cotyledons with stronger resistance were able to form a protective region of intensive oxidative bursts between the areas with and without hyphal intrusions to block further fungal advancement to the uninfected regions. The qPCR analysis suggested that different onset patterns of some marker genes in ROS accumulation/programmed cell death (PCD) such as RBOHD, MPK3 were associated with distinct levels of resistance from B. napus cultivars against L. maculans. The observations and datasets from this article indicated the distinct differences in ROS-related cellular behaviors and signaling between compatible and incompatible interactions.

NAPDH Oxidase-Specific Flow Cytometry Allows for Rapid Genetic Triage and Classification of Novel Variants in Chronic Granulomatous Disease.

PURPOSE: Chronic granulomatous disease (CGD) is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. Dihydrorhodamine (DHR) flow cytometry is the standard diagnostic test for CGD, and correlates with NADPH oxidase activity. While there may be genotype correlation with the DHR flow pattern in some patients, in several others, there is no correlation. In such patients, assessment by flow cytometric evaluation of NADPH oxidase-specific (NOX) proteins provides a convenient and rapid means of genetic triage, though immunoblotting has long been used for this purpose. METHODS AND RESULTS: We describe the clinical utility of the NOX flow cytometry assay through assessment of X-linked and autosomal recessive CGD patients and their first-degree relatives. The assessment of specific NOX proteins was correlated with overall NADPH oxidase function (DHR flow), clinical phenotype and genotype. NOX-specific protein assessment is a valuable adjunct to DHR assessment and genotyping to classify and characterize CGD patients. CONCLUSIONS: The atypical clinical presentation of some CGD patients can make genotype-phenotype correlation with DHR flow data challenging. Genetic testing, while useful for confirmation of diagnosis, can take several weeks, and in some patients does not provide a conclusive answer. However, NADPH-oxidase-specific protein flow assessment offers a rapid alternative to identification of the underlying genetic defect in cellular subsets, and can be utilized as a reflex test to an abnormal DHR flow. Further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes.

In silico insights on diverse interacting partners and phosphorylation sites of respiratory burst oxidase homolog (Rbohs) gene families from Arabidopsis and rice.

BACKGROUND: NADPH oxidase (Nox) is a critical enzyme involved in the generation of apoplastic superoxide (O2-), a type of reactive oxygen species (ROS) and hence regulate a wide range of biological functions in many organisms. Plant Noxes are the homologs of the catalytic subunit from mammalian NADPH oxidases and are known as respiratory burst oxidase homologs (Rbohs). Previous studies have highlighted their versatile roles in tackling different kind of stresses and in plant growth and development. In the current study, potential interacting partners and phosphorylation sites were predicted for Rboh proteins from two model species (10 Rbohs from Arabidopsis thaliana and 9 from Oryza sativa japonica). The present work is the first step towards in silico prediction of interacting partners and phosphorylation sites for Rboh proteins from two plant species. RESULTS: In this work, an extensive range of potential partners (unique and common), leading to diverse functions were revealed from interaction networks and gene ontology classifications, where majority of AtRbohs and OsRbohs play role in stress-related activities, followed by cellular development. Further, 68 and 38 potential phosphorylation sites were identified in AtRbohs and OsRbohs, respectively. Their distribution, location and kinase specificities were also predicted and correlated with experimental data as well as verified with the other EF-hand containing proteins within both genomes. CONCLUSIONS: Analysis of regulatory mechanisms including interaction with diverse partners and post-translational modifications like phosphorylation have provided insights regarding functional multiplicity of Rbohs. The bioinformatics-based workflow in the current study can be used to get insights for interacting partners and phosphorylation sites from Rbohs of other plant species.

BcIqg1, a fungal IQGAP homolog, interacts with NADPH oxidase, MAP kinase and calcium signaling proteins and regulates virulence and development in Botrytis cinerea.

NADPH oxidases (Nox) produce reactive oxygen species (ROS) in multicellular eukaryotic organisms. They trigger defense reactions ('oxidative burst') - in phagocytes and plant cells -, and are involved in a broad range of differentiation processes. Fungal Nox-complexes play a central role in vegetative, sexual and pathogenic processes. In contrast to mammalian systems, knowledge is limited about composition, localisation and connection to major signaling cascades in fungi. Here, we characterize a fungal homolog of the RasGAP scaffold protein IQGAP, which links several major signaling processes, including Nox in mammalian cell lines. We show that BcIqg1 interacts directly with a cytosolic, regulatory component (BcRac) and a membrane-associated subunit (BcNoxD) of a Nox-complex in the pathogen Botrytis cinerea. Thus, this protein may be a scaffold that mediates interaction of the catalytic subunits with the regulator BcNoxR. The protein interacts with modules of the MAP kinase- and calcium-dependent signaling pathways. Functional analysis of BcIqg1 substantiated its involvement in different signaling pathways. It mediates the Ca(2+) -triggered nuclear translocation of - BcCRZ1 and the MAP kinase BcBmp1. BcIqg1 is involved in resistance against oxidative and membrane stress and is required for several developmental processes including formation of sclerotia, conidial anastomosis tubes and infection cushions as well as for virulence.

A single nucleotide polymorphism in the NCF1 gene leading to reduced oxidative burst is associated with systemic lupus erythematosus.

OBJECTIVES: Ncf1 polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases in animal models. The human NCF1 gene is very complex with both functional and non-functional gene copies and genotyping requires assays specific for functional NCF1 genes. We aimed at investigating association and function of the missense single nucleotide polymorphism (SNP), rs201802880 (here denoted NCF1-339) in NCF1 with systemic lupus erythematosus (SLE). METHODS: We genotyped the NCF1-339 SNP in 973 Swedish patients with SLE and 1301 controls, using nested PCR and pyrosequencing. ROS production and gene expression of type 1 interferon-regulated genes were measured in isolated cells from subjects with different NCF1-339 genotypes. RESULTS: We found an increased frequency of the NCF1-339 T allele in patients with SLE, 11% compared with 4% in controls, OR 3.0, 95% CI 2.4 to 3.9, p=7.0×10-20. The NCF1-339 T allele reduced extracellular ROS production in neutrophils (p=0.004) and led to an increase expression of type 1 interferon-regulated genes. In addition, the NCF1-339 T allele was associated with a younger age at diagnosis of SLE; mean age 30.3 compared with 35.9, p=2.0×1-6. CONCLUSIONS: These results clearly demonstrate that a genetically controlled reduced production of ROS increases the risk of developing SLE and confirm the hypothesis that ROS regulate chronic autoimmune inflammatory diseases.

Efferocytosis is impaired in Gaucher macrophages.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylceramide-laden macrophages resulting from impaired digestion of aged erythrocytes or apoptotic leukocytes. Studies of macrophages from patients with type 1 Gaucher disease with genotypes N370S/N370S, N370S/L444P or N370S/c.84dupG revealed that Gaucher macrophages have impaired efferocytosis resulting from reduced levels of p67phox and Rab7. The decreased Rab7 expression leads to impaired fusion of phagosomes with lysosomes. Moreover, there is defective translocation of p67phox to phagosomes, resulting in reduced intracellular production of reactive oxygen species. These factors contribute to defective deposition and clearance of apoptotic cells in phagolysosomes, which may have an impact on the inflammatory response and contribute to the organomegaly and inflammation seen in patients with Gaucher disease.

Modern management of phagocyte defects.

Phagocytic neutrophil granulocytes are among the first immune cells active at sites of infection, forming an important first-line defense against invading microorganisms. Congenital immune defects concerning these phagocytes may be due to reduced neutrophil numbers or function. Management of affected patients depends on the type and severity of disease. Here, we provide an overview of causes and treatment of diseases associated with congenital neutropenia, as well as defects of the phagocytic respiratory burst.

Effect of recombinant bovine somatotropin on leukocyte mRNA expression for genes related to cell energy metabolism, cytokine production, phagocytosis, oxidative burst, and adaptive immunity.

Objectives of the current experiment were to evaluate the effects of treatment of periparturient dairy cows with recombinant bovine somatotropin (rbST) on mRNA expression in peripheral leukocytes for genes related to the somatotropic axis, cell energy metabolism, and innate and adaptive immune responses. Cows were enrolled in the experiment at 253 ± 3 d of gestation and assigned to an untreated control group (n = 98) or to receive 125 mg of rbST weekly from -21 to 21 d relative to calving (rbST125; n = 98). Data from a subsample of cows (control = 16, rbST125 = 16) are reported herein. Hemogram and polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of adhesion molecules were determined weekly, from -28 ± 3 to 24 ± 3 d relative to calving. Cows were vaccinated with ovalbumin at -21 ± 3, -7 ± 3, and 7 ± 3 d relative to calving. Serum IgG anti-ovalbumin and haptoglobin optical densities were determined weekly, from -28 ± 3 to 24 ± 3 d relative to calving. Leukocytes were isolated from whole blood on d -21 ± 3, -7 ± 3, and 7 ± 3 relative to calving to determine leukocyte mRNA expression for 66 genes. Cows in the rbST125 treatment had greater concentration of granulocytes 1 wk prepartum (control = 3.7 ± 0.4 vs. rbST125 = 4.6 ± 0.4 × 109 cells/L). Expression of CD18 by PMNL during the prepartum (control = 3,262 ± 280 vs. rbST125 = 3,926 ± 260 geometric mean fluorescence intensity) and percentage of PMNL positive for phagocytosis and oxidative burst 1 wk postpartum (control = 59.2 ± 2.8 vs. rbST125 = 67.6 ± 3.1%) were increased in rbST125 cows. Postpartum IgG anti-ovalbumin optical density was higher in rbST125 cows than control cows (control = 1.5 ± 0.1 vs. rbST125 = 1.9 ± 0.1 optical density). On d -7 relative to calving, leukocyte mRNA expression of IGF1R and JAK1 tended to be downregulated and expression of DEFB3 tended to be upregulated by rbST treatment. Expression of mRNA for STAT1, RELA, and NOD2 tended to be upregulated, expression of RAC2 was downregulated, and expression of JAK3, BLK, and TNFα tended to be downregulated on d 7 relative to calving by rbST treatment. Effects of treatment of periparturient cows with 125 mg of rbST on leukocyte gene expression were minute; thus, additional experiments are necessary to elucidate how rbST-induced increases in growth hormone and insulin-like growth factor-1 concentrations may modulate the immune response of periparturient cows.

Clinical, Immunological, and Molecular Findings of Patients with p47phox Defect Chronic Granulomatous Disease (CGD) in Indian Families.

Chronic granulomatous disease (CGD) is a group of inherited disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex. Reduced or absent oxygen radical synthesis seen in these patients leads to impaired killing of intracellular bacteria and fungi. CGD clinically presents with recurrent and life-threatening infections as well as granulomatous inflammatory responses. p47phox encoded by the NCF1 gene is the most common autosomal recessive form of CGD which is often clinically milder. Here, we are presenting the data on clinical and immunological findings in 21 Indian patients with Del GT mutation in the NCF1 gene. Diagnosis of these patients was based on detailed clinical evaluation, measurement of respiratory burst activity by nitro blue tetrazolium and dihydrorhodamine-1,2,3 assay, expression of p47phox by flow cytometry, and molecular confirmation by GeneScan method. Seventeen male and four female patients with median age of onset of 1 year ranging from 1.5 months to 6 years were included in the study. Sixty-two percent (13 out of 21) of patients belonged to a consanguineous marriage with only one family having a history of a previous sibling death. Significant variability in clinical presentation was observed in spite of identical genetic defect ranging from asymptomatic to very severe presentation leading to early death or requiring transplantation. However, none of these patients showed difference in immunological parameters to account for this variability. Thus, this study highlights the phenotypic heterogeneity seen in these patients with Del GT mutation in the NCF1 gene and its implication in management of these patients.

Dendritic cell immunoreceptor 1 alters neutrophil responses in the development of experimental colitis.

BACKGROUND: Ulcerative colitis, an inflammatory bowel disease, is associated with the massive infiltration of neutrophils. Although the initial infiltration of neutrophils is beneficial for killing bacteria, it is presumed that persistent infiltration causes tissue damage by releasing antibacterial products as well as inflammatory cytokines. A murine C-type lectin receptor, dendritic cell immunoreceptor 1 (Dcir1), is expressed on CD11b(+) myeloid cells, such as macrophages, dendritic cells and neutrophils. It was reported that Dcir1 is required to maintain homeostasis of the immune system to prevent autoimmunity, but it is also involved in the development of infectious disease resulting in the enhanced severity of cerebral malaria. However, the role of Dcir1 in intestinal immune responses during colitis remains unclear. In this study, we investigated the role of Dcir1 in intestinal inflammation using an experimental colitis model induced with dextran sodium sulfate (DSS). RESULTS: In contrast to wild type (WT) mice, Dcir1 (-/-) mice exhibited mild body weight loss during the course of DSS colitis accompanied by reduced colonic inflammation. Dcir1 deficiency caused a reduced accumulation of neutrophils in the inflamed colon on day 5 of DSS colitis compared with WT mice. Consistently, the production of a neutrophil-attracting chemokine, MIP-2, was also decreased in the Dcir1 (-/-) colon compared with the WT colon on day 5. There were fewer myeloperoxidase-positive neutrophils in the inflamed colon of Dcir1 (-/-) mice than in that of WT mice. Moreover, bone marrow neutrophils from Dcir1 (-/-) mice produced less reactive oxygen species (ROS) by lipopolysaccharide stimulation than those from WT mice. This suggests that Dcir1 deficiency decreases the accumulation of tissue destructive neutrophils during DSS colitis. CONCLUSION: Dcir1 enhances the pathogenesis of DSS colitis by altering neutrophil recruitment and their functions.

Monocyte/macrophage-specific NADPH oxidase contributes to antimicrobial host defense in X-CGD.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency disease that is characterized by susceptibility to bacterial and fungal infections. Various mutations in CYBB encoding the gp91(phox) subunit of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase impair the respiratory burst of all types of phagocytic cells and result in X-linked CGD (X-CGD). PURPOSE: We here sought to evaluate the underlying cause in an attenuated phenotype in an X-CGD patient. The patient is a 31-year-old male who had been diagnosed as having X-CGD based on the absence of nitroblue tetrazolium reduction and the presence of a CYBB mutation at the age of 1 year. He has been in good health after overcoming recurrent bacterial infections in infancy. METHODS: We investigated genomic DNA analysis of CYBB gene, residual activity of NADPH oxidase, and expression of gp91(phox) in both polymorphonuclear leukocytes (PMNs) and monocytes/macrophages in the present patient. RESULTS: Although his underlying germline mutation, c.1016C>A (p.P339H) in the CYBB gene, was identified in both PMNs and monocytes, the expression and functional activity of gp91(phox) retained in monocytes/macrophages, in stark contrast to markedly reduced PMNs. CONCLUSIONS: Our results indicate that residual reactive oxygen intermediates (ROI) production in PMNs plays an important role in infantile stage in X-CGD, but thereafter retained function of monocytes/macrophages might compensate for the function of NADPH oxidase deficient PMNs and might be an important parameter for predicting the prognosis of X-CGD patients.

Cell death control: the interplay of apoptosis and autophagy in the pathogenicity of Sclerotinia sclerotiorum.

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.

Impaired neutrophil function in 24p3 null mice contributes to enhanced susceptibility to bacterial infections.

Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens for which siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions, but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. In this study, we show that neutrophils from mice genetically deficient for lipocalin 24p3 (24p3(-/-)) are defective in many neutrophil functions. Specifically, neutrophils in 24p3(-/-) mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3(-/-) neutrophils. Additionally, small regulatory RNAs (microRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3(-/-) neutrophils. Further, 24p3(-/-) neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3(-/-) mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans and Staphylococcus aureus) pathogens. Listeria does not secrete siderophores, and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3(-/-) mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function.

Assembly of NADPH oxidase in human neutrophils is modulated by the opacity-associated protein expression State of Neisseria gonorrhoeae.

Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91(phox)/p22(phox)) and the cytoplasm (p47(phox), p67(phox), and p40(phox)). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa-) Gc does not stimulate neutrophil ROS production. Using constitutively Opa- and OpaD-positive (OpaD+) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa- Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD+ Gc. gp91(phox) and p22(phox) were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa- Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47(phox) "organizing" subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.

The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1.

The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins) that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR) gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular basis of the wheat-S. nodorum interaction, an emerging model for necrotrophic pathosystems.

Suppression of dendritic cell-mediated responses by genes in calcium and cysteine protease pathways during Mycobacterium tuberculosis infection.

With rising incidence of acquired drug resistance among life-threatening pathogens, alternative approaches to improve therapy and vaccination have taken center stage. To this end, genome-wide and pathway-specific siRNA libraries are being employed increasingly to identify genes that regulate immune responses against a number of pathogens. In this study using calcium and cysteine protease pathway-specific siRNA libraries, we identified genes that play critical roles in modulating diverse functions of dendritic cells (DCs) during Mycobacterium tuberculosis infection. Knockdown of many of these genes in the two pathways resulted in reduced bacterial burden within DCs. These included genes that regulated activation of transcription factors, ubiquitin-specific peptidases, and genes that are involved in autophagy and neddylation. Knockdown of certain genes increased the expression of IL-12p40 and surface densities of costimulatory molecules in an antigen- and receptor-specific manner. Increased IL-12p40 and costimulatory molecules on DCs also promoted the development of Th1 responses from a Th2 inducing antigen. Furthermore, modulation of autophagy and oxidative burst appeared to be one of the mechanisms by which these genes regulated survival of M. tuberculosis within DCs. Although some genes regulated specific responses, others regulated multiple responses that included IL-12 production, T cell priming, as well as intracellular survival of M. tuberculosis. Further dissection of the mechanisms such as neddylation, by which these genes regulate immune responses, would improve our understanding of host parameters that are modulated during M. tuberculosis infection.