Frank Ebel

Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus -induced activation of murine macrophages

Aspergillus fumigatius is a ubiquitous saprophytic fungus that has become the most prevalent airborne fungal pathogen for immunocompromised patients during the last two decades. In this report we have analysed how macrophages recognize this microorganism. Using transfected human HEK 293 cells we demonstrate that NF‐κB‐dependent promoter activation triggered by A. fumigatus is mediated by Toll‐like receptors TLR2 and TLR4, whereas no activation was observed in cells overexpressing other distinct TLR proteins (TLR1, TLR3, TLR5–10). Using macrophages derived from mice lacking TLR2 expression, expressing defective TLR4 or both we found that A. fumigatus conidia and hyphae induce NF‐κB translocation, release of pro‐inflammatory molecules, like TNFα, and the chemoattractant MIP‐2 in a TLR2‐ and TLR4‐dependent manner. Recognition of A. niger and A. fumigatus , was similar in terms of the parameters analysed, suggesting that pathogenic and non‐pathogenic aspergilli are sensed by macrophages in a similar fashion. Finally, we found that recruitment of neutrophils is severely impaired in mice lacking both functional TLR2 and TLR4, but is less impaired in single TLR2‐ or TLR4‐deficient mice, providing evidence that both receptors are required for an optimal immune response to Aspergillus in vivo.

Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C‐type lectins represent a family of receptors, which recognize pathogen‐specific carbohydrates. One of them is β1–3 glucan, a major component of the fungal cell wall. Here we provide evidence that β1–3 glucan plays an important role for the elimination of A.u2003fumigatus conidia. Laminarin, a soluble β1–3 glucan and antibodies to dectin‐1, a well known β1–3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible β1–3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of β1–3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild‐type conidia are phagocytosed with high efficiency thereby confirming the relevance of β1–3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2‐mediated recognition of A.u2003fumigatus and the phagocytic response.

NETs formed by human neutrophils inhibit growth of the pathogenic mold Aspergillus fumigatus

Neutrophil extracellular traps (NETs) represent a distinct mechanism to control and eliminate microbial infections. Our results show that conidia and germ tubes of the human pathogenic mold Aspergillus fumigatus are able to trigger the formation of NETs. Viable fungal cells are not essentially required for this host-pathogen interaction. Neutrophils engulf conidia and thereby inhibit their germination, a process that is independent of NETosis. In the experimental set-up used in this study neutrophils do not kill germ tubes, but reduce their polar growth and this inhibition depends on NETs as it can be overcome by the addition of DNase-1. The Zn(2+) chelator calprotectin is associated with the Aspergillus-induced NETs and addition of Zn(2+) abrogates the NET-mediated growth inhibition. In summary, our data provide evidence that NETs are not sufficient to kill A. fumigatus, but might be a valuable tool to confine infection.

Aspergillus fumigatus: contours of an opportunistic human pathogen

Aspergillus fumigatus is currently the major air‐borne fungal pathogen. It is able to cause several forms of disease in humans of which invasive aspergillosis is the most severe. The high mortality rate of this disease prompts increased efforts to disclose the basic principles of A. fumigatus pathogenicity. According to our current knowledge, A. fumigatus lacks sophisticated virulence traits; it is nevertheless able to establish infection due to its robustness and ability to adapt to a wide range of environmental conditions. This review focuses on two crucial aspects of invasive aspergillosis: (i) properties of A. fumigatus that are relevant during infection and may distinguish it from non‐pathogenic Aspergillus species and (ii) interactions of the pathogen with the innate and adaptive immune systems.

Lactoferrin impairs type III secretory system function in enteropathogenic Escherichia coli

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. EPEC uses a type III secretory system to deliver effector proteins into the host cell. These proteins cause the characteristic attaching and effacing lesion on enterocytes. Lactoferrin, a glycoprotein present in human milk, inhibits EPEC adherence to mammalian cells. To determine the effect of lactoferrin on the initial host cell attachment step that is mediated by the type III secretory system, we focused on EPEC-induced actin polymerization in HEp2 cells, on the hemolytic activity, and on measurement of E. coli secreted proteins A, B, and D (EspABD). Lactoferrin blocked EPEC-mediated actin polymerization in HEp2 cells and blocked EPEC-induced hemolysis. The mechanism of this inhibition was lactoferrin-mediated degradation of secreted proteins necessary for bacterial contact and pore formation, particularly EspB. The proteolytic effect of lactoferrin was prevented by serine protease inhibitors. This disruption of the type III secretory system implies that lactoferrin could provide broad cross protection against the enteropathogens that share this mechanism.

Human NK Cells Display Important Antifungal Activity against Aspergillus fumigatus, Which Is Directly Mediated by IFN-γ Release

Despite the strong interest in the NK cell-mediated immunity toward malignant cells and viruses, there is a relative lack of data on the interplay between NK cells and filamentous fungi, especially Aspergillus fumigatus, which is the major cause of invasive aspergillosis. By studying the in vitro interaction between human NK cells and A. fumigatus, we found only germinated morphologies to be highly immunogenic, able to induce a Th1-like response, and capable of upregulating cytokines such as IFN-γ and TNF-α. Moreover, priming NK cells with human rIL-2 and stimulating NK cells by direct NK cell–pathogen contact were essential to induce damage against A. fumigatus. However, the most interesting finding was that NK cells did not mediate anti-Aspergillus cytotoxicity through degranulation of their cytotoxic proteins (perforin, granzymes, granulysine), but via an alternative mechanism involving soluble factor(s). To our knowledge, our study is the first to demonstrate that IFN-γ, released by NK cells, directly damages A. fumigatus, attributing new properties to both human NK cells and IFN-γ and suggesting them as possible therapeutic tools against IA.

Farnesol misplaces tip‐localized Rho proteins and inhibits cell wall integrity signalling in Aspergillus fumigatus

Farnesol is known for inducing apoptosis in some fungi and mammalian cells. To evaluate its potential role as an antifungal agent, we studied its impact on the human pathogen Aspergillus fumigatus. We found that growth of A. fumigatus wild type is inhibited, but two cell wall mutants, Δmnt1 andΔglfA, are much more susceptible to farnesol. This susceptibility is partially rescued by osmotic stabilization, suggesting that farnesol is a cell wall perturbing agent. However, farnesol does not activate but inhibit the cell wall integrity (CWI) pathway. Remarkably, mutants lacking AfMkk2 or AfMpkA, two kinases essential for CWI signalling, are also highly susceptible to farnesol, suggesting that its mode of action goes beyond inhibition of CWI signalling. Farnesyl derivatives are known for interfering with the function of prenylated proteins. We analysed the subcellular localization of two prenylated Rho family GTPases, AfRho1 and AfRho3, which are implicated in controlling CWI and the cytoskeleton. We found that under normal growth conditions AfRho1 and AfRho3 predominantly localize to the hyphal tip. After farnesol treatment this localization is rapidly lost, which is accompanied by swelling of the hyphal tips. Parallel displacement of tropomyosin from the tips suggests a concomitant disorganization of the apical actin cytoskeleton.

Analysis of the major proteins secreted by the human opportunistic pathogen Aspergillus fumigatus under in vitro conditions

Although secreted proteins of pathogenic microorganisms often represent potential virulence factors, so far only limited information has been available on the proteins secreted by Aspergillus fumigatus. We therefore analysed supernatant proteins after growth in different media. In serum-free cell culture medium A. fumigatus growth was limited and no protein secretion was detectable, whereas distinct protein patterns were detectable after growth in either aspergillus minimal medium (AMM) or the more complex yeast glucose medium (YG). The three major proteins secreted under these conditions were identified as the ribotoxin mitogillin, a chitosanase and the aspergillopepsin i. Mitogillin and chitosanase were secreted in AMM, whereas aspergillopepsin i was especially prominent after growth in YG. When the AMM cultures reached stationary phase, seven additional major proteins were detectable. Two of them were identified as the chitinase chiB1 and a beta(1-3) endoglucanase. Conditioned medium containing mitogillin and chitosanase did not have a detectable cytotoxic effect on A549 and Vero cells. Using recombinant mitogillin and chitosanase we detected anti-chitosanase and antimitogillin antibodies in sera of patients suffering from invasive aspergillosis or aspergilloma, but not in control sera of healthy individuals. This suggests that chitosanase, like mitogillin, is expressed during infection and might therefore be of diagnostic relevance.

Extrapulmonary Aspergillus infection in patients with CARD9 deficiency

Invasive pulmonary aspergillosis is a life-threatening mycosis that only affects patients with immunosuppression, chemotherapy-induced neutropenia, transplantation, or congenital immunodeficiency. We studied the clinical, genetic, histological, and immunological features of 2 unrelated patients without known immunodeficiency who developed extrapulmonary invasive aspergillosis at the ages of 8 and 18. One patient died at age 12 with progressive intra-abdominal aspergillosis. The other patient had presented with intra-abdominal candidiasis at age 9, and developed central nervous system aspergillosis at age 18 and intra-abdominal aspergillosis at age 25. Neither patient developed Aspergillus infection of the lungs. One patient had homozygous M1I CARD9 (caspase recruitment domain family member 9) mutation, while the other had homozygous Q295X CARD9 mutation; both patients lacked CARD9 protein expression. The patients had normal monocyte and Th17 cell numbers in peripheral blood, but their mononuclear cells exhibited impaired production of proinflammatory cytokines upon fungus-specific stimulation. Neutrophil phagocytosis, killing, and oxidative burst against Aspergillus fumigatus were intact, but neither patient accumulated neutrophils in infected tissue despite normal neutrophil numbers in peripheral blood. The neutrophil tissue accumulation defect was not caused by defective neutrophil-intrinsic chemotaxis, indicating that production of neutrophil chemoattractants in extrapulmonary tissue is impaired in CARD9 deficiency. Taken together, our results show that CARD9 deficiency is the first known inherited or acquired condition that predisposes to extrapulmonary Aspergillus infection with sparing of the lungs, associated with impaired neutrophil recruitment to the site of infection.

Role of Respiration in the Germination Process of the Pathogenic Mold Aspergillus fumigatus

Inhalation of resting conidia is usually the first step of a systemic infection caused by the opportunistic fungal pathogen Aspergillus fumigatus. In the lung, the inhaled spores encounter an environment that permits germination. However, the relative importance of certain environmental conditions for conidial activation and subsequent hyphae formation has so far not been analyzed in detail. In this study, we studied the role of oxygen during germination. We found that inhibitors of the respiratory chain were nearly as efficient in blocking germination as cycloheximide, an inhibitor of protein synthesis, which is already known to prevent germination of Aspergillus nidulans. We also found that A. fumigatus is unable to grow or germinate under anaerobic conditions, and using the fluorescent mitotracker dye we detected active mitochondria already at the stage of swollen conidia, which indicates that respiration is an early event during germination. In line with these data, we found that significant oxygen consumption was detectable early during germination, whereas no oxygen consumption was measurable in suspensions of resting conidia. In summary, the present study provides evidence that respiration is absolutely required for the germination of A. fumigatus conidia.