Surface Structure Characterization of Aspergillus fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response

In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface.     

Stability indicating RP-LC determination of sildenafil citrate (Viagra) in pure form and in pharmaceutical samples

A simple, precise, sensitive and stability-indicating reverse-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitation of sildenafil citrate (SC) in pure form and its pharmaceutical formulations. Method employs water and acetonitrile (48:52 v/v) as mobile phase with flow rate of 1 ml min(-1), LiChrospher C18-5 microm (25 x 0.46 cm) column and UV detection set at 245 nm. The internal standard method using piroxicam (PX) as the internal standard is used. The linear dynamic range of SC was found to be 0.05-7.5 microg ml(-1). The proposed method is successfully employed for the determination of SC in the tablets. The excipients present in the formulations do not interfere with the assay procedure. Analytical parameters were calculated and full statistical evaluation is included.     

Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins

Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 μg/ml; voriconazole MIC, 2 μg/ml; ravuconazole MIC, 4 μg/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 μg/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the β-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazole-resistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.     

Aspergillus fumigatus LaeA-Mediated Phagocytosis Is Associated with a Decreased Hydrophobin Layer

Aspergillus fumigatus is the causal agent of the life-threatening disease invasive aspergillosis. A. fumigatus laeA deletants, aberrant in toxin biosynthesis and spore development, are decreased in virulence. Among other characteristics, the decreased virulence is associated with increased spore susceptibility to macrophage phagocytosis. Three characteristics, cell wall microbe-associated molecular patterns (MAMPs), secreted metabolites, and rodlet content, thought to be important in macrophage-Aspergillus spore interactions were examined. Flow cytometry analysis of wild-type and ΔlaeA spores did not reveal any differences in surface-accessible MAMPs, including β-(1,3)-glucan, α-mannose, chitin, and other carbohydrate ligands. Blocking experiments with laminarin and mannan supported the conclusion that differences in cell wall carbohydrates were not responsible for enhanced ΔlaeA spore phagocytosis. Aspergillus spores have been reported to secrete metabolites affecting phagocytosis. Neither spent culture exchange, transwell, nor coincubation internalization experiments supported a role for secreted metabolites in the differential uptake of wild-type and ΔlaeA spores. However, sonication assays implicated a role for surface rodlet protein/hydrophobin (RodAp) in differential spore phagocytosis. A possible role of RodAp in enhanced ΔlaeA spore uptake was further assessed by RodAp extraction and quantification, where wild-type spores were found to contain 60% more RodAp than ΔlaeA spores. After removal of the surface rodlet layer, wild-type spores were phagocytosed at similar rates as ΔlaeA spores. We conclude that increased uptake of ΔlaeA resting spores is not associated with changes in secreted metabolite production of this mutant or surface carbohydrate availability but, rather, due to a decrease in the surface RodAp content of ΔlaeA spores. We theorize that RodAp acts as an antiphagocytic molecule, possibly via physicochemical means and/or by impeding MAMP recognition by macrophage receptors.Infectious fungal diseases are an increasingly intractable cause of mortality worldwide. The most common filamentous agents of disease are members of the genus Aspergillus. These species are primarily saprophytic molds that can produce a wide spectrum of diseases in both plants and animals. The species responsible for more than 90% of human disease is A. fumigatus (7, 12, 15, 18). Disease symptoms run the spectrum from allergic bronchopulmonary aspergillosis to invasive aspergillosis (IA), the latter particularly on the rise owing to increased numbers of immunocompromised patients, including those afflicted with HIV/AIDS, malignancy, and organ dysfunction. High aspergillosis mortality rates emphasize the need to find improved means to treat these diseases. Our overall goal is to develop a better understanding of both fungal and host mechanisms contributing to disease development as a platform toward developing enhanced protective therapies.The internalization of spores by macrophages is a key component of the early immune defense against fungal infections, including Aspergillus infections. A compilation of studies of spore phagocytosis has presented a partial understanding of this complex process. Recognition of the spore is a key requirement for both the phagocytosis of the spore and the subsequent activation of macrophages. Surface molecules, now termed microbe-associated molecular patterns (MAMPs) (8), are critical for recognition of spores. The best-characterized fungal MAMP is β-(1,3)-glucan, recognized by the mammalian receptor dectin-1 (5). Low amounts of surface-accessible β-(1,3)-glucans on resting spores of A. fumigatus are associated with decreased phagocytosis indexes compared to swollen spores, which display increased amounts of surface β-(1,3)-glucans (13). Similarly, increased internalization of resting spores of the A. fumigatus pksP mutant (lacking spore pigments) is associated with higher concentrations of surface β-(1,3)-glucans (11, 13).In addition to β-(1,3)-glucan recognition, other molecules have been speculated to play a role in spore phagocytosis. Although not well defined, metabolites diffused from spores have been reported to affect macrophage handling of A. fumigatus spores (14), including inhibition of phagocytosis (2). Spore architecture influences cellular internalization as well; the outermost cell wall layer of Aspergillus spores is decorated with interwoven proteinaceous microfibrils called rodlets. These are hydrophobins encoded primarily by RODA that confer physiochemical properties to mediate spore dispersal and, possibly, cellular interactions (9, 20). In particular ΔrodA mutants, lacking the rodlet layer, are more sensitive to macrophage killing (16). Spore pigments, encoded by developmentally regulated clustered genes including pksP, confer resistance to macrophage phagocytosis and intracellular killing (10, 21-23), seemingly through enhanced β-(1,3)-glucan availability as discussed above (13). Null mutants of the dioxygenase PpoC, showing altered spore morphology, are also taken up more readily by macrophages, although the mechanism underlying this increased internalization has not been uncovered.Previously, we reported on an A. fumigatus mutant, ΔlaeA, that displayed decreased virulence. Among other characteristics, decreased virulence was associated with increased susceptibility to macrophage phagocytosis. Spores harvested from ΔlaeA strains produced diminished amounts of at least one metabolite in vitro, and surface features of spores harvested from ΔlaeA strains showed an overall loss of prominent protrusions, which was speculated to be associated with abnormalities in rodlet formation (3). Here, our goal was to elucidate fungal modifications in ΔlaeA conidia related to phagocytosis. We provide evidence that increased uptake of ΔlaeA resting spores is not associated with changes in secreted metabolite production of this mutant nor with β-(1,3)-glucan (or other surface carbohydrate) availability but, rather, is associated with decreased RodAp content of ΔlaeA spores.     

Impaired phagocytosis directs human monocyte activation in response to fungal derived β‐glucan particles

Recognition of the fungal cell wall carbohydrate β‐glucan by the host receptor Dectin‐1 elicits broad immunomodulatory responses, such as phagocytosis and activation of oxidative burst. These responses are essential for engulfing and killing fungal pathogens. Phagocytic monocytes are key mediators of these early host inflammatory responses to infection. Remarkably, whether phagocytosis of fungal β‐glucan leads to an inflammatory response in human monocytes remains to be established. Here, we show that phagocytosis of heat‐killed Candida albicans is essential to trigger inflammation and cytokine release. By contrast, inhibition of actin‐dependent phagocytosis of particulate (1‐3,1‐6)‐β‐glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal β‐glucan particles upon actin cytoskeleton disruption, relies on Dectin‐1 and results in the classical caspase‐1 inflammasome formation through NLRP3, generation of an oxidative burst, NF‐κB activation, and increased inflammatory cytokine release. PI3K and NADPH oxidase were crucial for both cytokine secretion and ROS generation, whereas Syk signaling mediated only cytokine production. Our results highlight the mechanism by which phagocytosis tightly controls the activation of phagocytes by fungal pathogens and strongly suggest that actin cytoskeleton dynamics are an essential determinant of the host's susceptibility or resistance to invasive fungal infections.     

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.     

Invasive aspergillosis in liver transplant recipients

Background

Liver transplantation is increasing worldwide with underlying pathologies dominated by metabolic and alcoholic diseases in developed countries.

Methods

We provide a narrative review of invasive aspergillosis (IA) in liver transplant (LT) recipients. We searched PubMed and Google Scholar for references without language and time restrictions.

Results

The incidence of IA in LT recipients is low (1.8%), while mortality is high (∼50%). It occurs mainly early (<3 months) after LT. Some risk factors have been identified before (corticosteroid, renal, and liver failure), during (massive transfusion and duration of surgical procedure), and after transplantation (intensive care unit stay, re-transplantation, re-operation). Diagnosis can be difficult and therefore requires full radiological and clinicobiological collaboration. Accurate identification of Aspergillus species is recommended due to the cryptic species, and susceptibility testing is crucial given the increasing resistance of Aspergillus fumigatus to azoles. It is recommended to reduce the dose of tacrolimus (50%) and to closely monitor the trough level when introducing voriconazole, isavuconazole, and posaconazole. Surgery should be discussed on a case-by-case basis. Antifungal prophylaxis is recommended in high-risk patients. Environmental preventative measures should be implemented to prevent outbreaks of nosocomial aspergillosis in LT recipient units.

Conclusion

IA remains a very serious disease in LT patients and should be promptly sought and, if possible, prevented by clinicians when risk factors are identified.