Norcal Spotted and BS Thread

@Tee Arghhhhhh Dee Well how's the ride?

 

Nah, Richmond

 

@Gply @Tee Arghhhhhh Dee

I think you both owe us all flex pictures still!

 

ABSTRACT
Summary: Aspergillus species are globally ubiquitous saprophytes found in a variety of ecological niches. Almost 200 species of aspergilli have been identified, less than 20 of which are known to cause human disease. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis (IA) in the immunocompromised patient population. IA is a devastating illness, with mortality rates in some patient groups reaching as high as 90%. Studies identifying and assessing the roles of specific factors of A. fumigatus that contribute to the pathogenesis of IA have traditionally focused on single-gene deletion and mutant characterization. In combination with recent large-scale approaches analyzing global fungal responses to distinct environmental or host conditions, these studies have identified many factors that contribute to the overall pathogenic potential of A. fumigatus. Here, we provide an overview of the significant findings regarding A. fumigatus pathogenesis as it pertains to invasive disease.

Go to:
INTRODUCTION
Aspergillus species are ubiquitous, saprophytic fungi that play a significant role in global carbon and nitrogen recycling. Although their primary ecological niche is soil or decaying vegetation, aspergilli produce small, hydrophobic conidia that disperse easily into the air and can survive a broad range of environmental conditions. The genus Aspergillus, which includes almost 200 species, has a tremendous impact on public health both beneficially as the workhorse of industrial applications and negatively as plant and human pathogens (71). Several Aspergillus species are utilized for their rich enzymatic profile in the industrial production of foods and pharmaceuticals. For example, Aspergillus niger is used for the industrial production of citric acid, amylases, pectinases, phytases, and proteases; A. terreus is used for the cholesterol-lowering drug lovastatin; and A. oryzae is used for the fermentation of soybeans and rice into soy sauce and sake, respectively. Aspergilli also have a less reputable side in the agricultural industry. Aspergillus section Flavi, particularly A. flavus and A. parasiticus, can contaminate several common crops with aflatoxin, a highly toxic carcinogen with immunosuppressive properties (228, 230). The consumption of contaminated crops can cause serious illness or death and is a common problem in developing countries.

The Human Pathogen A. fumigatus
Among the human pathogenic species of Aspergillus, A. fumigatus is the primary causative agent of human infections, followed by A. flavus, A. terreus, A. niger, and the model organism, A. nidulans (54, 135). Aspergilli cause a wide range of human ailments depending on the immune status of the host (54, 107). In individuals with altered lung function such as asthma and cystic fibrosis patients, aspergilli can cause allergic bronchopulmonary aspergillosis, a hypersensitive response to fungal components. Noninvasive aspergillomas may form following repeated exposure to conidia and target preexisting lung cavities such as the healed lesions in tuberculosis patients. Invasive aspergillosis (IA) is perhaps the most devastating of Aspergillus-related diseases, targeting severely immunocompromised patients. Those most at risk for this life-threatening disease are individuals with hematological malignancies such as leukemia; solid-organ and hematopoietic stem cell transplant patients; patients on prolonged corticosteroid therapy, which is commonly utilized for the prevention and/or treatment of graft-versus-host disease in transplant patients; individuals with genetic immunodeficiencies such as chronic granulomatous disease (CGD); and individuals infected with human immunodeficiency virus (54, 97, 126, 133, 148, 162, 227). Mortality rates range from 40% to 90% in high-risk populations and are dependent on factors such as host immune status, the site of infection, and the treatment regimen applied (114). The severity and increased incidence of IA necessitate a better understanding of the interplay between host and fungus that contributes to A. fumigatus pathogenesis (130). Pathogenesis and virulence are terms used here in the context of altered host immune function, as this organism is inherently an opportunistic pathogen, and disease pathology and progression are the result of both fungal growth and the host response. In this review, we will thus discuss the pathogenic potential of A. fumigatus as a progression of the infectious life cycle within the context of these immunodeficiencies.

Invasive Aspergillosis


Infectious life cycle.
Aspergilli are predominantly saprophytes, growing on dead or decaying matter in the environment. The infectious life cycle of Aspergillus begins with the production of conidia (asexual spores) that are easily dispersed into the air, ensuring ubiquity in both indoor and outdoor environments (Fig. (Fig.1)1) (65, 137). The primary route of human infection is via the inhalation of these airborne conidia, followed by conidial deposition in the bronchioles or alveolar spaces. In healthy individuals, conidia that are not removed by mucociliary clearance encounter epithelial cells or alveolar macrophages, the primary resident phagocytes of the lung. Alveolar macrophages are primarily responsible for the phagocytosis and killing of Aspergillusconidia as well as the initiation of a proinflammatory response that recruits neutrophils (one type of polymorphonuclear cell [PMN]) to the site of infection. Conidia that evade macrophage killing and germinate become the target of infiltrating neutrophils that are able to destroy hyphae. The risk of developing IA results primarily from a dysfunction in these host defenses in combination with fungal attributes that permit A. fumigatus survival and growth in this pulmonary environment (176). Although other host responses have been associated with disease resistance, for this review, we will focus on fungal interactions with the primary innate components that are most important for fungal defense.


FIG. 1.
Infectious life cycle of A. fumigatus. Aspergillus is ubiquitous in the environment, and asexual reproduction leads to the production of airborne conidia. Inhalation by specific immunosuppressed patient groups results in conidium establishment in the ...


Risk factors and pathology.
The primary host immunodeficiencies that are responsible for the increased risk of IA are neutropenia and corticosteroid-induced immunosuppression, and the pathological consequences of IA under these immunosuppressive conditions differ, as described previously for patients and animal models (9, 17, 53, 192). Prolonged neutropenia is classically defined as the most dominant risk factor for IA and is often the result of highly cytotoxic therapies such as cyclophosphamide, which is used for transplant patients or those with hematological diseases. Cyclophosphamide, a DNA-alkylating agent, binds to DNA and interferes with cellular replication, depleting circulating white blood cells including neutrophils. In neutropenic patients and animal models of chemotherapy-induced neutropenia, IA is characterized by thrombosis and hemorrhage from rapid and extensive hyphal growth (41, 192). The lack of inflammatory infiltrates, despite the production of tumor necrosis factor alpha (TNF-α), results in low levels of inflammation. Without neutrophil recovery, angioinvasion and dissemination to other organs via the blood result.

A variety of nonneutropenic patients, most commonly those on corticosteroid therapy such as allogeneic transplant patients receiving corticosteroids for prophylaxis or treatment of graft-versus-host disease, are susceptible to IA, although the pathology of the disease is quite different. IA in these patients and nonneutropenic animal models is nonangioinvasive, characterized by limited fungal development with pyogranulomatous infiltrates, tissue necrosis, and excessive inflammation. Corticosteroids have significant consequences for phagocyte function, including but not limited to the impairment of phagocytosis, phagocyte oxidative burst, production of cytokines and chemokines, and cellular migration (reviewed in reference 116). Several studies have shown that corticosteroids impair the functional ability of phagocytes to kill A. fumigatus conidia and hyphae (37, 92, 132, 171, 172, 214). Despite the effects of steroids on innate immune cell function, neutrophils are recruited to the lung and prevent hyphal invasion but create an inflammatory environment that results in tissue injury. This exacerbated inflammatory response is generally regarded as being the cause of death, in contrast to the uncontrolled fungal growth observed in neutropenic hosts. The dramatic differences in both fungal development and host responses under each immunosuppressive regimen highlight the importance of studying Aspergillus pathogenesis within the context of host immune status and subsequent response to fungal infection.

Animal Models of Invasive Aspergillosis
Identification of the contribution of individual fungal components to overall pathogenicity requires the use of in vivo models of IA. Drosophila melanogaster (104, 115, 186) and Galleria mellonella (140, 163, 164, 166) have been applied to screen A. fumigatus mutants for virulence attributes owing to their ethical and financial advantages over the use of mammalian models. However, results should be interpreted with caution, and interesting phenotypes should be reevaluated using a more applicable animal model. For example, the difference in temperature (flies and worms, which are unable to grow at 37°C, are grown at 25°C) is known to affect multiple fungal characteristics including growth rate and toxin production (see below), and clearly, these models cannot be used to assess pathological outcomes of infection that are relevant to human infection. Indeed, a recent study highlights the need for caution in using Galleria; those authors found that melanin mutants known to be less virulent in mammalian studies (see below) were more virulent in the G. mellonellamodel (86). A variety of vertebrates including rats, rabbits, birds, and guinea pigs have been used, but mouse models predominate due to the availability of genetically defined species and reagents (42). Outbred mice are commonly chosen because of their cost compared to that of inbred strains, but sufficient numbers should be used to establish reproducibility due to the inherent genetic variability within populations. On the other hand, although inbred mice offer the advantage of genetic reproducibility, studies between individual inbred strains can be vastly different, such that comparisons of multiple inbred strains may benefit studies of fungal pathogenesis.

Specific genetic mouse models exist. CGD (p47phox−/−) or X-CGD (gp91phox−/−) mice display pathological consequences (such as peribronchiolar and alveolar necrosis) of A. fumigatus infection similar to those for humans with CGD and have been a useful model for studying aspergillosis in the context of this specific genetic disease (136, 161). The importance of pattern recognition receptors (PRRs) to fungal recognition and modulation of host responses has been clarified with the use of knockout mice, such as those for dectin-1 and several of the Toll-like receptors (TLRs) (142). Cytokine-deficient mice have also been used to demonstrate the contribution of cytokines to host resistance (such as TNF-α) or susceptibility (interleukin-10 [IL-10]) (33, 40, 158).

The most commonly used animal models of IA involve the induction of neutropenia or corticosteroid-induced immunosuppression to mimic human infection. Neutropenia may be induced by cyclophosphamide or other chemotherapeutic agents (antibody-mediated neutrophil depletion has also been used), whereas animals treated with corticosteroids represent the nonneutropenic model used to evaluate A. fumigatuspathogenesis in the context of inflammatory responses commonly observed in nonneutropenic patients. The use of specific drug or depletion regimens is known to influence survival, pathology, and other outcome parameters (191). Comparison of both models can help to differentiate the fungus-host interactions responsible for pathogenesis in unique patient populations. One of the most striking examples of this is in the case of gliotoxin mutants, which demonstrate wild-type virulence in a neutropenic model but reduced virulence in a nonneutropenic model, suggesting that gliotoxin may be important for pathogenicity only in the context of nonneutropenic hosts (Table (Table11).


TABLE 1.
Comparison of gliotoxin mutants in different animal models of IA
Other variables to account for when establishing an appropriate animal model to assess fungal pathogenesis include the amount of fungal inoculum, route of infection, and outcome analyses (58). Conidial inoculation may be performed intratracheally, intranasally, intravenously, or via inhalation chamber. Intranasal inoculation is commonly used because of ease of handling, although chamber inhalation is potentially the most useful model in terms of both reproducibility and mimicking human infection (182, 190). Outcome analyses often chosen for assessing disease development include animal survival, organ pathology, host cellular responses, and fungal burden, all of which can be influenced by the variables described above (191).

The interaction of fungi with mammalian cells in vitro can be a useful complement to in vivo studies and can steer experiments toward the appropriate in vivo assays. For example, although a ΔgliZ gliotoxin mutant displayed virulence similar to that of a wild-type strain in the neutropenic mouse model, gliotoxin production did contribute to neutrophil apoptosis in vitro, supporting the observed virulence reduction of gliotoxin mutants in a nonneutropenic model and reduced neutrophil apoptosis at sites of infection (25, 186, 197). In vitro studies with primary mammalian cells and cell lines are frequently used to assess the role of specific fungal components during fungus-host cell interactions, although A. fumigatus mutants that display altered interactions with host cells in vitro do not always correlate with virulence defects in vivo, particularly when the only in vivo assessment made is animal mortality. This is in agreement with the multifactorial nature of A. fumigatus pathogenesis and emphasizes the significance of examining other outcomes of infection, such as histological analyses or fungal burden. Minor contributions of fungal components to overall pathogenicity may thus be characterized by studying the interaction of A. fumigatus with mammalian cells with further investigation in vivo to understand the pathogenesis of this disease. The use and careful interpretation of animal models and outcomes, as well as in vitro host systems, are thus essential to study the role of fungal and host elements in a disease setting that mimics human infection.

Biology of Aspergillus fumigatus
A genomic comparison of clinical and environmental isolates from diverse host sources and geographic locations suggested that any environmental strain of A. fumigatus may be pathogenic given an appropriate host (50). In comparison to other species, A. fumigatus displays a unique combination of basic traits that contribute to pathogenicity. The primary route of infection with Aspergillus is via the inhalation of airborne conidia and deposition in the bronchioles or alveolar spaces. The average size of A. fumigatus conidia (2 to 3 μm) is ideal for infiltrating deep into the alveoli, whereas larger conidia of other human pathogens including A. flavus and A. niger could be removed more easily by mucociliary clearance of the upper respiratory tract. Furthermore, A. fumigatus is more thermotolerant than other disease-causing species, growing well at 37°C and withstanding temperatures above 50°C, such as those encountered in decaying vegetation, a frequently inhabited niche. It has been speculated that growth at high temperatures may induce the expression of unique stress response genes that confer additional virulence benefits, although evidence for this theory is lacking.

Several studies suggested that the radial growth and germination rate of aspergilli at 37°C correlate with pathogenicity. The deletion of A. fumigatus genes involved in morphogenesis, including the regulatory subunit of the cyclic AMP-dependent protein kinase signaling gene pkaR and the ras family subhomologue rasB, resulted in reduced germination and growth rates in vitro, correlating with reduced virulence in a murine model of IA (67, 235). Mutants of the calcineurin pathway, which is involved in cellular stress responses and morphogenesis, are significantly impaired in growth, exhibiting defects in conidial germination and polarized hyphal growth at 37°C, and are significantly impaired in causing disease in multiple animal models of IA (48). Additionally, a comparison of A. fumigatus, A. flavus, and A. niger growths demonstrated a correlation between the germination rate and pathogenic prevalence. The germination rates of these species were similar at temperatures up to 30°C but differed at 37°C and 42°C (8). Interestingly, human sera, specifically albumin, enhance mycelial growth of several Aspergillus species in vitro and specifically promote conidial germination in A. fumigatus (170). It would appear, therefore, that one factor contributing to the pathogenicity of A. fumigatus is growth rate in vivo, specifically at 37°C. Indeed, the deletion of a gene involved in ribosome biogenesis, cgrA, in A. fumigatus had no effect on growth at 25°C or virulence in a Drosophila insect model (25°C) but was slower in radial growth at 37°C and was reduced in virulence in an animal model (37°C) (19, 23). These studies correlate the rate of growth at 37°C with virulence.

Go to:
AIRWAY COLONIZATION
Inhalation of Aspergillus conidia is a common occurrence due to their ubiquitous presence in the environment; estimates suggest that the average person may inhale up to 200 conidia per day. In IA-susceptible patient populations, the mucosal defenses of the lung are compromised, leading to fungal colonization and growth.

Aspergillus Interactions with Soluble Lung Components
Following inhalation, A. fumigatus conidia immediately encounter the airway mucosa comprised of the fluid lining the respiratory tract and airway epithelia. This pulmonary fluid is comprised of mucus, proteins, lipids, ions, water, and other cellular secretions that contribute to the mucociliary clearance of inhaled particles or pathogens. Also within this complex fluid are opsonic PRRs that coat inhaled pathogens and contribute to host defense. Among these proteins are the collectins, a group of C-type lectin receptors secreted by type II cells and Clara cells that bind carbohydrate moieties in a calcium-dependent manner. Many pathogenic fungi, including A. fumigatus, have a carbohydrate-rich cell wall that can be recognized by the most common collectins, mannose-binding lectin (MBL) and the surfactant proteins SP-A and SP-D. In vitro, MBL, SP-A, and SP-D have been shown to bind and agglutinate A. fumigatus conidia as well as enhance the phagocytosis and killing of A. fumigatus conidia by macrophages and neutrophils (2, 3, 120, 144). MBL, SP-A, and SP-D were more recently found to activate complement (59). Collectins have been demonstrated to be important in vivo: MBL−/− and SP-D−/− mice exhibit increased susceptibility to IA, and recombinant MBL, SP-D, and SP-A have been used to enhance host defenses against aspergillosis in animal models (94, 121). Collectins may thus contribute to conidial clearance by enhancing complement activation, phagocytosis, and killing of conidia or aggregating conidia for other host defenses.

One of the earliest host responses to microorganisms is the activation of complement, a collection of serum proteins that recognize and bind conserved microbial constituents, resulting in opsonization or destruction. Although found predominantly in serum, complement components are present, albeit at lower levels, in bronchoalveolar fluid and have the potential to be involved in host defense against Aspergillus. Three complement pathways exist, converging at binding of C3 to the microbial surface. Early studies demonstrated that A. fumigatus conidia and hyphae bind C3. In comparison to other human pathogenic species, A. fumigatus as well as A. flavus bind fewer C3 molecules per unit of conidial surface (75). The majority of bound C3 is cleaved to iC3b, a ligand for phagocytic complement receptors; thus, A. fumigatus and A. flavusmay be less susceptible to complement-mediated phagocytosis or phagocyte recognition (193, 194). Conidia and hyphae from several Aspergillus species also bind the alternative complement inhibitor factor H, its splice product FHR-1, and factor H family protein FFHL-1, preventing the activation of complement cascades (13, 213). Binding to the classical and lectin pathway inhibitor C4b-binding protein has been observed for A. fumigatus (213). Finally, A. fumigatus and A. flavus, but not A. niger, were found to produce a soluble complement-inhibitory factor, potentially lipid derived, that prevented alternative pathway activation (219, 221). It would thus appear that A. fumigatus and, to a lesser-known extent, A. flavus have defense mechanisms to inhibit or reduce complement activation. Thus, the ability of A. fumigatus to inhibit complement activation may contribute to the overall pathogenesis of this organism.

Other soluble components involved in Aspergillus defense include the pentraxin PTX3 and plasminogen. PTX3 is a soluble opsonin produced by phagocytes that facilitates microbial recognition (28). Mice deficient in PTX3 are susceptible to IA, which correlated with a reduced recognition of A. fumigatus conidia by phagocytes (69). A recent study implicated genetic variation in plasminogen, a component of the fibrolytic pathway, in susceptibility to IA (233). Plasminogen bound to A. fumigatus has also been detected, which, when cleaved into active plasmin, could enhance dissemination via its role in the degradation of the extracellular matrix (13).

Aspergillus Interaction with Respiratory Epithelia
Despite the importance of respiratory epithelia in initiating antimicrobial innate immune responses against many inhaled pathogens, few studies have examined the role of the airway epithelia in the host defense against Aspergillus (12, 127). As the first cells encountered by inhaled conidia, airway epithelia likely contribute to the overall immune response to A. fumigatus. Epithelial cells may secrete soluble antimicrobial compounds that play a direct role in airway defense. Members of the defensin family of antimicrobial peptides have broad-spectrum activity against multiple microbes and are produced by epithelial cells in vitro following incubation with A. fumigatus (1). In vitro, Aspergillus germinating conidia and hyphae, but not resting conidia, are recognized by host PRRs on epithelial cells and induce the production of cytokines and chemokines such as IL-6, TNF-α, and IL-8 (10, 14). Corticosteroid administration can eliminate this inflammatory response, questioning the function of epithelial cells in corticosteroid-treated patients at risk for IA (14). Epithelial cells likely assist in initiating proinflammatory responses against A. fumigatus, although their contribution is likely far less robust than that of the alveolar macrophage.

A. fumigatus conidia have been shown to bind and be engulfed by a variety of epithelial cells including tracheal epithelial cells, alveolar type II cells, human nasal epithelial cells, and the A549 lung epithelial cell line (66, 155, 223). Conidia engulfed by A549 epithelia enter acidic phagolysosomes and can be killed, although some conidia are able to germinate and exit both the phagolysosome and pneumocyte without evidence of pneumocyte damage (222, 223). A. fumigatus conidia are also able to inhibit drug- or TNF-α-induced apoptosis in primary epithelial cells and epithelial cell lines in vitro, although the in vivo implications of this are unknown (18).

Several fungally derived factors may contribute to the ability of A. fumigatus to bind and modulate the airway epithelium (Fig. (Fig.2).2). One factor contributing to A. fumigatus binding and uptake by epithelial cells is the presence of sialic acid residues on conidia (34, 51). Interestingly, pathogenic species of aspergilli, including A. fumigatus, display more conidial sialic acid than do nonpathogenic aspergilli (224). Adhesion to fibrinogen, the basement membrane glycoprotein laminin, and the extracellular matrix component fibronectin is also partially mediated by sialic acid residues and other proteins on the conidial surface (7, 30, 31, 34, 46, 202, 203). The significance of conidial binding to these components is linked to the fact that lung injury or distress is a risk factor for IA. Fibrinogen (fibrin after processing) and fibronectin attach to wounded surfaces such as the surface of damaged epithelia, and laminin is exposed upon epithelial injury or detachment. Sialic acid, and perhaps other conidial factors that bind to alveolar components, could thus contribute to pathogenicity by enhancing adhesion to and colonization of epithelia and components of injured tissue.


FIG. 2.
Interaction of A. fumigatus with respiratory epithelia. Following inhalation, A. fumigatus encounters airway epithelia (lining trachea, bronchi, and bronchioles), the mucus and fluid lining the upper respiratory tract, and, ultimately, the alveolar space. ...
A. fumigatus may facilitate colonization in otherwise healthy lung tissue via secreted products that alter epithelial function and viability. Culture filtrates from A. fumigatus strains have been shown to induce cell shrinkage, desquamation, and actin cytoskeleton rearrangement in A549 cells (93, 98). The activity of filtrates could be inhibited with serine and cysteine protease inhibitors, implicating protease activity. Indeed, when a specific serine protease, AF-ALF, was deleted, culture filtrates failed to induce actin cytoskeleton damage (98). Protease activity in A. fumigatus culture filtrates has also been linked to human nasal epithelial cell detachment and loss of focal contacts that may assist germinating hyphae in invading the lung tissue (98, 169, 201).

Human respiratory epithelial cell damage and slowed ciliary beat frequency from A. fumigatus culture filtrates and sputum samples obtained from patients with pulmonary aspergillosis have been linked to secondary metabolites, specifically gliotoxin and, at higher concentrations, fumagillin and helvolic acid (5, 6, 43). The tremorigenic metabolite verruculogen, produced in conidial and hyphal filtrates of many A. fumigatus strains, has also been implicated in modifying transepithelial resistance, hyperpolarization, and cytoplasmic vacuolization of human nasal epithelial cells in an air-liquid interface model of the airway epithelium (29, 96). Associated with conidia and hyphal elements, verruculogen could impact the airway epithelium during early infection, although production in vivo has yet to be observed (96). Thus, A. fumigatus is able to interfere with mucociliary clearance, bind respiratory epithelia and basement membrane proteins, and invade or damage epithelial cells to establish infection and potentially evade other host defenses.

Aspergillus and the Alveolar Macrophage


Macrophage responses to Aspergillus.
Alveolar macrophages are the primary resident phagocytic cells of the respiratory tract and a critical component of the host defense against Aspergillus conidia. Alveolar macrophages phagocytose Aspergillus conidia in an actin-dependent manner, a process mediated by the recognition of pathogen-associated molecular patterns by host cell PRRs. PRR engagement of A. fumigatusligands generates a proinflammatory response characterized by the production of cytokines and chemokines that are important for host defense against this organism, including TNF-α, IL-1β, IL-6, IL-8, macrophage inflammatory protein 1α, and monocyte chemoattractant protein 1 (39, 131, 138, 158). TLR2 and TLR4 and the C-type lectin receptor dectin-1 are the most well-characterized PRRs involved in the recognition of A. fumigatus and the activation of host cells. In vitro studies have demonstrated that conidia and hyphae activate macrophages through TLR2 and TLR4, and TLR2 recognizes both conidial and hyphal morphologies, whereas TLR4 recognizes only the hypha form (143). Studies using TLR−/− mice also suggest an essential role for TLR4, and potentially TLR2, in vivo. In neutropenic models, TLR2−/− and TLR4−/− mice exhibit higher fungal burdens than wild-type mice (11, 15). Although TLR4−/− mice have lower survival rates than wild-type mice in these studies, contradicting results were demonstrated for TLR2−/− mice. The role of TLRs in nonneutropenic models has not been well studied, although TLR4 polymorphisms in allogeneic hematopoietic stem cell transplant patients have been associated with an increased risk for IA (22).

Unlike TLRs, dectin-1 is essential for the host defense against Aspergillus in both immunosuppressed and immunocompetent hosts (226). Dectin-1 is specific for the fungal carbohydrate β(1,3)-glucan, which is normally masked on resting A. fumigatus conidia by the proteinaceous hydrophobin layer. Following conidial swelling, β(1,3)-glucan becomes exposed and is present on swollen conidia, germlings, and hypha morphotypes (70, 77). Dectin-1-β(1,3)-glucan engagement results in phagocytosis, macrophage activation, and a strong induction of proinflammatory responses (35, 70, 77, 119, 189, 210). Thus, dectin-1, with additional contributions by TLRs, enables innate immune cells to phagocytose and kill conidia as well as elicit proinflammatory responses.

Alveolar macrophages kill conidia that have swollen within the phagolysosome with reactive oxygen species (ROS) and phagolysosomal acidification (83, 159, 220). Philippe et al. first demonstrated the role of ROS in macrophage-mediated conidial killing. In these experiments, alveolar macrophages from p47phox−/− CGD mice, or wild-type alveolar macrophages treated with chemical inhibitors of NADPH oxidase, were significantly impaired in their ability to kill A. fumigatus conidia (159). Although other studies suggested that NADPH-mediated oxidative responses do not contribute to alveolar macrophage killing of conidia, several factors (macrophage-to-conidium ratios, coincubation times, and animal strains and/or cell types tested) can lead to conflicting results (136, 177). It does appear that nonoxidative mechanisms contribute to conidial killing. Reactive nitrogen species are ineffective, although other candidates (antimicrobial peptides, for example) have yet to be tested (159). Overall, immunosuppressed patients who are susceptible to IA have reduced alveolar macrophage effector functions, either from corticosteroid-mediated suppression or from chemotherapy-induced depletion, resulting in the ability of A. fumigatus to escape macrophage killing.



A. fumigatus defenses. (i) Melanin.
In addition to masking β(1,3)-glucan and delaying macrophage activation, resting A. fumigatus conidia are resistant to macrophage killing (Fig. (Fig.3).3). The protective role of the pigment melanin against host defenses, specifically via scavenging ROS, has been described for many pathogenic fungi (87, 106). In Aspergillus, melanin provides the conidial pigment that has been used to distinguish between some species. In A. fumigatus, a white, pigmentless strain was first described by Jahn et al. following UV mutagenesis (88). Complementation of the wild-type phenotype using an A. fumigatuscosmid library identified a polyketide synthase gene, pksP, as being the source of pigment production (105). The white mutant displayed ultrastructural cell wall differences and increased susceptibility to the oxidants H2O2 and NaOCl in comparison to wild-type conidia (88). Additionally, white conidia induced greater monocyte and neutrophil production of ROS than did the wild type as a result of wild-type melanin scavenging ROS from the culture medium. Monocytes were able to kill more ingested mutant conidia than wild-type conidia, presumably via ROS-mediated mechanisms. In an animal model of systemic IA, the white conidia were less virulent, demonstrating for the first time the direct role of melanin in A. fumigatuspathogenesis. These studies implicate melanin as being an important contributor to pathogenesis as an ROS scavenger. It should be noted that the systemic animal model of IA does not fully recapitulate the infectious process of IA, as conidia are instilled directly into the blood via the tail vain as opposed to intranasally or intratracheally. Clearly, this administration could lead to a very different host response to A. fumigatus. Studies of another melanin mutant in an intranasal model (see below), however, support the role for melanin in A. fumigatus pathogenesis. One study also implicated the involvement of the cyanide-insensitive alternative oxidase in protecting conidia from macrophage ROS-mediated killing (the hyphal component not being explored) (123). In that study, however, RNA interference was used to knock down aox expression in a melanin mutant such that alternative oxidase may be important for protection against macrophage killing only in the context of a melanin deficiency.


FIG. 3.
A. fumigatus interactions with phagocytes. Alveolar macrophages phagocytose inhaled conidia via PRRs. Conidial swelling (within or outside of the macrophage) releases the protective rodlet layer, exposing β(1,3)-glucan for recognition by dectin-1. ...
Additional studies indicated that human macrophage engulfment of pksP mutant conidia resulted in an increased acidification of the phagosome as a result of phagolysosomal fusion (89). The addition of chloroquine, which increases phagolysosomal fusion and pH, resulted in wild-type killing similar to that of the mutant. A functional pksP therefore prevented some level of phagolysosomal fusion, increasing conidial survival. Furthermore, that study implicated phagolysosome acidification as being a mechanism of conidial killing.

The initial discovery of pksP coincided with the identification by Tsai et al. and Watanabe et al. of another gene involved in pigment biosynthesis, arp1, a naphthopyrone synthase and homologue of scytalone dehydratase (206, 225). Mutants of arp1 were reddish-pink and bound fewer C3 molecules than wild-type conidia, implicating melanin or a pigmented intermediate synthesized by arp1 in the defense against host complement. Further study of conidial pigment biosynthesis in A. fumigatus identified a six-gene cluster involved in dihydroxynaphthalene-melanin biosynthesis, including pksP (also called alb1) (207). The deletion of alb1, similar to the observations by Jahn et al., led to a white phenotype and reduced virulence in mice (205). The alb1 mutants were also more susceptible to C3 binding, reinforcing the notion that melanin or melanin intermediates may prevent complement activation. Another melanin, pyomelanin, produced by the tyrosine degradation pathway, may also contribute to pathogenicity, as mutants showed an enhanced sensitivity to ROS (178). Overall, melanin appears to be a significant determinant of A. fumigatuspathogenesis by protecting conidia against multiple host defenses, particularly those of the alveolar macrophage.



(ii) Mediators of ROS defense.
Other molecules implicated in pathogenicity as scavengers of toxic ROS include rodlets and superoxide dismutases (SODs). Conidia are surrounded by a hydrophobic layer comprised of rodlet proteins. Of the two rodlet genes in A. fumigatus, rodA is solely responsible for rodlet production, and rodA mutants display increased susceptibility to alveolar macrophage killing (156). Rodletless conidia also induced a weak inflammatory response in a rat model of IA (183). Given that conidial swelling is essential for macrophage activation, the rodletless conidia may induce more rapid and robust macrophage activation and conidial elimination. The two eukaryotic SOD enzymes, Cu/Zn-SOD and Mn-SOD, have not been well studied for their role in A. fumigatus pathogenesis. Cu/Zn-SOD has been detected in the cell wall of conidia and hyphae, and SOD activity in culture filtrates has been demonstrated (73, 78). Although Cu/Zn-SOD is upregulated under low-iron conditions in vitro and sera from some patients with A. fumigatus infections react with Cu/Zn-SOD, a specific role in vivo has yet to be identified (38, 73, 79). McDonagh et al. found increased levels of Mn-SOD transcripts in response to neutrophils in vitro and during infection in a murine model of IA, implicating this SOD in oxidative stress defense (129).

Studies of rare hypervirulent mutants substantiate the contribution of oxidative stress resistance to pathogenicity. A disruption of the α(1,3)-glucan synthase gene ags3 led to increased virulence in an animal model of IA, correlating with resistance to oxidative stress in vitro, perhaps related to the increased melanin content or increased germination rate observed in this mutant (128). An unrelated mutant of oxylipin biosynthesis, in which the three cyclooxygenase genes in A. fumigatus were silenced by RNA interference, also demonstrated increased virulence correlating with increased resistance to H2O2 (208). The individual deletion of the cyclooxygenase genes did not yield an increase in virulence, although a loss of one gene, ppoC, resulted in greater resistance to H2O2 as well as aberrant conidium morphology and increased phagocytosis by macrophages (49). Finally, the deletion of the glycosylphosphatidylinositol-anchored protein ECM33 resulted in a hypervirulent strain with increased germination and resistance to cell wall-destabilizing agents (174). Intriguingly, the deletion of the ace2 gene, encoding a C2H2 zinc finger transcription factor, resulted in altered conidial pigmentation, cell wall organization, and resistance to H2O2 (63). The ace2mutant was hypervirulent in a nonneutropenic mouse model but not in a neutropenic model, implicating resistance to neutrophil oxidative defenses. Transcripts of ags3, ecm33, and ppoC in this mutant were also examined and found to be lower than those of the wild type, suggesting that ACE2 regulation of these genes may contribute to some of the phenotypic similarities among these mutants.

 

What. A. Dick.

 

I just learned something about TW.

We can't make a post over 60,000 characters long.

 

Thankfully. Your last ramblings were beginning to look like those of a volcom type

 

Get out.

 

Rides like a caddy!!! It's a little stiff still (the ride) but I've only driven like 5 miles tops so far. Definitely a huge improvement, but I need to get it out on some dirt and see how it feels. I need to take out the extra leaf in the back, Phil's pack definitely put it on its tippy toes. So far, curbs and speed bumps are no problem.

 

Get it aligned yet?

 

Cant find anywhere around my house to run a wheel up on. I'll have to drive it to work this one day this week

 

Yup, yesterday. Forgot the specs though, they're in the glovebox. Everything looked and felt good. Definitely feels a little different without the sway bar on there, or it could just be I'm not used to the suspension yet.

 

Didnt think he was firing shots at me. He was obviously saying the other dude is a chump, which may be true. Before someone fucks with the dudes livelihood, I "felt the need" to say that the "chump" made it right as a professional should. Enjoy the show, keyboard warrior. You appear to be the only chump around here, anyway.

 

Indeed. I was obviously pretty mad at the time. I can't say I would never do something like that, lord knows I'm full of stupid ideas.

 

TV;DR?

 

Those on and off ramps take a little getting use. As @EDDO said its that initial lunge that'll give the pucker factor the first few times. I don't even notice it anymore. And having 700's helps also.