Uncoupling of IL-6 signaling and LC3-associated phagocytosis drives immunoparalysis during sepsis.

Immune deactivation of phagocytes is a central event in the pathogenesis of sepsis. Herein, we identify a master regulatory role of IL-6 signaling on LC3-associated phagocytosis (LAP) and reveal that uncoupling of these two processes during sepsis induces immunoparalysis in monocytes/macrophages. In particular, we demonstrate that activation of LAP by the human fungal pathogen Aspergillus fumigatus depends on ERK1/2-mediated phosphorylation of p47phox subunit of NADPH oxidase. Physiologically, autocrine IL-6/JAK2/Ninein axis orchestrates microtubule organization and dynamics regulating ERK recruitment to the phagosome and LC3+ phagosome (LAPosome) formation. In sepsis, loss of IL-6 signaling specifically abrogates microtubule-mediated trafficking of ERK, leading to defective activation of LAP and impaired killing of bacterial and fungal pathogens by monocytes/macrophages, which can be selectively restored by IL-6 supplementation. Our work uncovers a molecular pathway linking IL-6 signaling with LAP and provides insight into the mechanisms underlying immunoparalysis in sepsis.

Aspergillus fumigatus conidial metalloprotease Mep1p cleaves host complement proteins.

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus, one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores (i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host.

Penetration of the Human Pulmonary Epithelium by Aspergillus fumigatus Hyphae.

Background: The airway epithelium is the first barrier interacting with Aspergillus fumigatus conidia after their inhalation, suggesting that this structure functions as point of entry of this fungus to initiate pulmonary aspergillosis. Methods: To study epithelial entry by A fumigatus, primary human reconstituted pseudostratified epithelium cultured in air-liquid interface as well as bronchial epithelial cell monolayers were infected with conidia. Results: Under these experimental conditions, we found that A fumigatus hyphae traversed the bronchial epithelium through a mechanism involving the recruitment of actin, which formed a tunnel that allows hyphae to enter the cells without disturbing their integrity. Conclusions: These findings describe a new mechanism by which A fumigatus hyphae penetrate the airway epithelial barrier and can infect its human host.

Pathway of glycine betaine biosynthesis in Aspergillus fumigatus.

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD(+) to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.

Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins.

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.

CD4(+) T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease.

Aspergillus fumigatus is a model fungal pathogen and a common cause of infection in individuals with the primary immunodeficiency chronic granulomatous disease (CGD). Although primarily considered a deficiency of innate immunity, CGD is also linked to dysfunctional T cell reactivity. Both CD4(+) and CD8(+) T cells mediate vaccine-induced protection from experimental aspergillosis, but the molecular mechanisms leading to the generation of protective immunity and whether these mechanisms are dysregulated in individuals with CGD have not been determined. Here, we show that activation of either T cell subset in a mouse model of CGD is contingent upon the nature of the fungal vaccine, the involvement of distinct innate receptor signaling pathways, and the mode of antigen routing and presentation in DCs. Aspergillus conidia activated CD8(+) T cells upon sorting to the Rab14(+) endosomal compartment required for alternative MHC class I presentation. Cross-priming of CD8(+) T cells failed to occur in mice with CGD due to defective DC endosomal alkalinization and autophagy. However, long-lasting antifungal protection and disease control were successfully achieved upon vaccination with purified fungal antigens that activated CD4(+) T cells through the endosome/lysosome pathway. Our study thus indicates that distinct intracellular pathways are exploited for the priming of CD4(+) and CD8(+) T cells to A. fumigatus and suggests that CD4(+) T cell vaccination may be able to overcome defective antifungal CD8(+) T cell memory in individuals with CGD.

Galactofuranose attenuates cellular adhesion of Aspergillus fumigatus.

Galactofuranose (Galf) is a major molecule found in cell wall polysaccharides, secreted glycoproteins, membrane lipophosphoglycans and sphingolipids of Aspergillus fumigatus. The initial step in the Galf synthetic pathway is the re-arrangement of UDP-galactopyranose to UDP-Galf through the action of UDP-galactopyranose mutase. A mutant lacking the AfUGM1 gene encoding the UDP-galactopyranose mutase has been constructed. In the mutant, though there is a moderate reduction in the mycelial growth associated with an increased branching, it remains as pathogenic and as resistant to cell wall inhibitors and phagocytes as the wild-type parental strain. The major phenotype seen is a modification of the cell wall surface that results in an increase in adhesion of the mutants to different inert surfaces (glass and plastic) and epithelial respiratory cells. The adhesive phenotype is due to the unmasking of the mannan consecutive to the removal of galactofuran by the ugm1 mutation. Removal of the mannan layer from the mutant surface by a mannosidase treatment abolishes mycelial adhesion to surfaces.