The Pathogenesis of Aspergillus fumigatus,Host Defense Mechanisms,and the Development of AFMP4 Antigen as a Vaccine

Aspergillus fumigatus is one of the ubiquitous fungi with airborne conidia, which accounts for most aspergillosis cases. In immunocompetent hosts, the inhaled conidia are rapidly eliminated. However, immunocompromised or immunodeficient hosts are particularly vulnerable to most Aspergillus infections and invasive aspergillosis (IA), with mortality from 50% to 95%. Despite the improvement of antifungal drugs over the last few decades, the therapeutic effect for IA patients is still limited and does not provide significant survival benefits. The drawbacks of antifungal drugs such as side effects, antifungal drug resistance, and the high cost of antifungal drugs highlight the importance of finding novel therapeutic and preventive approaches to fight against IA. In this article, we systemically addressed the pathogenic mechanisms, defense mechanisms against A. fumigatus, the immune response, molecular aspects of host evasion, and vaccines’ current development against aspergillosis, particularly those based on AFMP4 protein, which might be a promising antigen for the development of anti-A. fumigatus vaccines.     

Azole Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in Patients with Cystic Fibrosis: A Matter of Concern?

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.     

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

Background

The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.

Methodology/Principal Findings

Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH₂) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH₂ supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.

Conclusions/Significance

The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.     

Chemosensitization prevents tolerance of Aspergillus fumigatus to antimycotic drugs

Tolerance of human pathogenic fungi to antifungal drugs is an emerging medical problem. We show how strains of the causative agent of human aspergillosis, Aspergillus fumigatus, tolerant to cell wall-interfering antimycotic drugs become susceptible through chemosensitization by natural compounds. Tolerance of the A. fumigatus mitogen-activated protein kinase (MAPK) mutant, sakAΔ, to these drugs indicates the osmotic/oxidative stress MAPK pathway is involved in maintaining cell wall integrity. Using deletion mutants of the yeast, Saccharomyces cerevisiae, we first identified thymol and 2,3-dihydroxybenzaldehyde (2,3-D) as potent chemosensitizing agents that target the cell wall. We then used these chemosensitizing agents to act as synergists to commercial antifungal drugs against tolerant strains of A. fumigatus. Thymol was an especially potent chemosensitizing agent for amphotericin B, fluconazole or ketoconazole. The potential use of natural, safe chemosensitizing agents in antifungal chemotherapy of human mycoses as an alternative to combination therapy is discussed.     

Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. Here, we present studies leading to the production of a set of red- and green-emitting luciferase mutants with bioluminescent properties suitable for expanding the use of the P. pyralis system to dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Using a combination of mutagenesis methods, we determined that the Ser284Thr mutation was sufficient to create an excellent red-emitting luciferase with a bioluminescence maximum of 615 nm, a narrow emission bandwidth, and favorable kinetic properties. Also, we developed a luciferase, containing the changes Val241Ile, Gly246Ala, and Phe250Ser, whose emission maximum was blue-shifted to 549 nm, providing a set of enzymes whose bioluminescence maxima were separated by 66 nm. Model studies demonstrated that in assays using a set of optical filters, the luciferases could be detected at the attomole level and seven orders of magnitude higher. In addition, in the presence of the Ser284Thr enzyme serving as a control, green light emission could be measured over a 10,000-fold range. The results presented here with the P. pyralis mutants provide evidence that simultaneous multiple analyte assay development is feasible with these novel proteins that require only a single substrate.     

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole–resistant strains of A. fumigatus. The TR₃₄/L98H and TR₄₆/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR₃₄/L98H resistance allele was the most common in all regions except South America where the TR₄₆/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.     

Biofilm Formation and its Impact on Antifungal Therapy

Fungi can protect themselves from host defences and antifungal drugs by the production of an extracellular hydrophobic matrix. Candida biofilms exhibit resistance to antifungal agents from all classes including the azoles, echinocandins, amphotericin B complex, and flucytosine. Although demonstrated on polystyrene and bronchial epithelia cells, until today, only indirect evidence for A. fumigatus biofilms in patients is available. The antifungals with the most activity against biofilms are the liposomal formulation of amphotericin B and agents in the echinocandin drug class. Importantly, echinocandins show excellent anti-biofilm activity against C. albicans at therapeutic concentrations. However, other biofilms formed by moulds, including A. fumigatus, are relatively resistant to echinocandins. Multiple mechanisms contribute to the intrinsic and acquired antifungal resistance during the different stages of fungal biofilm development. During the growth phase of the early biofilm various factors account for biofilm resistance. Combinational and sequential antifungal therapy as well as combination with enhancers can improve the effect of a single drug. Further studies are warranted to develop new therapeutic strategies targeting fungal biofilm-specific resistance mechanisms.     

Contribution of galactofuranose to the virulence of the opportunistic pathogen Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.     

In vitro interaction between glabridin and voriconazole against Aspergillus fumigatus isolates

BackgroundVoriconazole (VRC) is widely recommended as the first-line therapy for invasive aspergillosis. However, surveillance studies have demonstrated that there is an increase in the frequency of azole resistance among Aspergillus fumigates isolates. In recent years, more studies on effective synergisms between natural agents and antifungal drugs have been published.AimsTo evaluate the synergistic antifungal effect of glabridin (Gla) and VRC against A. fumigatus isolates.MethodsPotential interactions between Gla and VRC were studied by using a microdilution checkerboard method based on the CLSI reference technique. To assess the interaction of drugs the fractional inhibitory concentration index (FICI) was calculated based on the Loewe Additivity model.ResultsThe minimum inhibitory concentrations (MIC) obtained with Gla alone were relatively high (MIC₅₀ 16 μg/ml). However, our results showed synergistic interaction between Gla and VRC against A. fumigatus strains, with FICI range values between 0.15 and 0.5.ConclusionsSynergistic activity of Gla and VRC against both VRC-sensitive and -resistant A. fumigatus isolates may lead to design new antifungal agents, especially for inhibiting those azole-resistant strains.     

Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

Update on Antifungal Resistance and its Clinical Impact

Candida and Aspergillus species are important causes of opportunistic infection in an ever-growing number of vulnerable patients, and these infections are associated with high mortality. This has partly been attributed to the emerging resistance of pathogenic fungi to antifungal therapy, which potentially compromises the management of infected patients. Multi-azole resistance of Aspergillus fumigatus is a current health problem, as well as is the co-resistance of Candida glabrata to both azoles and echinocandins. In most cases, negative clinical consequences of reduced in vitro fungal susceptibility to azoles and/or echinocandins can be traced to acquisition of particular resistance mechanisms. While strategies using antifungal combinations or adjunctive agents that maximize the efficacy of existing antifungals may limit treatment failures, new therapeutic approaches, including antifungal agents with novel mechanisms of action, are urgent. In the meantime, more efforts should be devoted to close monitoring of antifungal resistance and its evolution in the clinical setting.     

Emerging Antifungal Drug Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and Among Other Species of <Emphasis Type="Italic">Aspergillus</Emphasis>

Purpose of Review

The purpose of this review is to give an overview of recent findings on antifungal resistance in Aspergillus fumigatus (the major causative agent of aspergillosis) and sibling Aspergillus species, which can be hidden agents of aspergillosis.

Recent Findings

Azole resistance by Cyp51A mutation in A. fumigatus is a growing problem worldwide. The resistance can occur in patients or in the environment. The former occurs by drug selection in the host, inducing mutations in Cyp51A. The latter is characterized by a tandem repeat in the promoter region of cyp51A gene and mutation(s) in Cyp51A. Environmental resistant strains are prevailing rapidly and globally. Moreover, efflux pump and biofilm formation are closely related with antifungal resistance of A. fumigatus. Finally, sibling species of Aspergillus are described with regard to antifungal resistance.

Summary

Environmental azole-resistant strains have newly emerged and been dispersed globally, and continuous survey and countermeasures are urgently needed against these strains. Although the contributions of Cyp51A and efflux pumps to antifungal resistance are becoming clear, other resistance mechanisms remain unclear. Further investigations including genome comparisons will help to clarify the novel resistant mechanisms and to develop countermeasures or novel antifungal drugs against resistant strains of A. fumigatus and other Aspergillus species that have low susceptibility to antifungal therapeutics.

     

Renal abscess due to Aspergillus fumigatus as the only sign of disseminated aspergillosis in a patient with AIDS

BackgroundAspergillus fumigatus can cause a wide variety of clinical syndromes, especially in the three largest immunocompromised groups, such as HIV-infected patients. Primary renal aspergillosis is an extremely rare entity.AimsWe report an unusual case of renal abscess due to Aspergillus fumigatus in a patient with AIDS.MethodsWe review clinical and laboratory records, and provide follow up of the patient.ResultsA 38-year-old man, HIV seropositive, was admitted to our hospital with fever, lumbar pain and respiratory symptoms. Abdominal ultrasound and computerised tomography showed a single and large lesion consistent with an abscess located in the left kidney. Aspergillus fumigatus was isolated from clinical sample obtained by ultrasound-guided needle aspiration. Despite a correct treatment based on amphotericin B and drainage of the abscess, surgery was necessary and nephrectomy was carried out. Histopathological examination of the surgical specimen confirmed the diagnosis of renal aspergillosis. Systemic antifungal therapy based on intravenous and oral voriconazole and highly active antiretroviral therapy was started after surgery. The patient had a good response to the established treatment and he remains in a good clinical condition at one year of follow up.ConclusionsCombined medical and surgical treatment is the elective therapy for renal abscesses due to Aspergillus when percutaneous drainage and the administration of systemic antifungal drugs, such as amphotericin B and/or oral voriconazole or itraconazole, fail. This case emphasizes renal fungal infections should be included in the differential diagnosis of kidney abscesses in AIDS patients.     

Aiming for a bull’s-eye: Targeting antifungals to fungi with dectin-decorated liposomes

Globally, there are several million individuals with life-threatening invasive fungal diseases such as candidiasis, aspergillosis, cryptococcosis, Pneumocystis pneumonia (PCP), and mucormycosis. The mortality rate for these diseases generally exceeds 40%. Annual medical costs to treat these invasive fungal diseases in the United States exceed several billion dollars. In addition to AIDS patients, the risks of invasive mycoses are increasingly found in immune-impaired individuals or in immunosuppressed patients following stem cell or organ transplant or implantation of medical devices. Current antifungal drug therapies are not meeting the challenge, because (1) at safe doses, they do not provide sufficient fungal clearance to prevent reemergence of infection; (2) most become toxic with extended use; (3) drug-resistant fungal isolates are emerging; and (4) only one new class of antifungal drugs has been approved for clinical use in the last 2 decades. DectiSomes represent a novel design of drug delivery to drastically increase drug efficacy. Antifungals packaged in liposomes are targeted specifically to where the pathogen is, through binding to the fungal cell walls or exopolysaccharide matrices using the carbohydrate recognition domains of pathogen receptors. Relative to untargeted liposomal drug, DectiSomes show order of magnitude increases in the binding to and killing of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus in vitro and similarly improved efficacy in mouse models of pulmonary aspergillosis. DectiSomes have the potential to usher in a new antifungal drug treatment paradigm.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.     

Micafungina en el tratamiento de la infección fúngica en modelos animales

BackgroundMicafungin is an echinocandin antifungal drug recently approved for the treatment of candidiasis. The possibility of its clinical use against other invasive mycoses, has aroused the interest of numerous investigators in evaluating its efficacy in different animal models.ObjectivesTo critically review the current data on the use of micafungin in the treatment of invasive mycoses in animal models.MethodsWe searched the PubMed/Medline data base (National Library of Medicine) from 2005 to 2008, both inclusive, on the use of micafungin in the experimental treatment of the fungal infection.Results and conclusionsSeven, of a total of 18 articles reviewed, were done in animal models of candidiasis and six in animal models of pulmonary or SNC aspergillosis. Similarly to the other echinocandins, caspofungin and anidulafungin, micafungin seems to exert a fungicidal activity against Candida albicans and Candida glabrata and a fungistatic activity against Aspergillus fumigatus. The paradoxical effect observed in lung tissue the experimental caspofungin treatment of aspergillosis has not been seen in the case of micafungin. The available data demonstrate a higher efficacy of micafungin versus fluconazole in the experimental treatment of C. albicans infections caused by strains susceptible in vitro to both drugs. To improve the efficacy of micafungin in the treatment of C. glabrata and A. fumigatus infections, several authors have tested different combined therapies, the combination of micafungin with amphotericin B being that showed the best results.     

Photolyase Confers Resistance to UV Light but Does Not Contribute to the Symbiotic Benefit of Bioluminescence in Vibrio fischeri ES114

Recent reports suggest that the selective advantage of bioluminescence for bacteria is mediated by light-dependent stimulation of photolyase to repair DNA lesions. Despite evidence for this model, photolyase mutants have not been characterized in a naturally bioluminescent bacterium, nor has this hypothesis been tested in bioluminescent bacteria under natural conditions. We have now characterized the photolyase encoded by phr in the bioluminescent bacterium Vibrio fischeri ES114. Consistent with Phr possessing photolyase activity, phr conferred light-dependent resistance to UV light. However, upon comparing ES114 to a phr mutant and a dark ΔluxCDABEG mutant, we found that bioluminescence did not detectably affect photolyase-mediated resistance to UV light. Addition of the light-stimulating autoinducer N-3-oxo-hexanoyl homoserine lactone appeared to increase UV resistance, but this was independent of photolyase or bioluminescence. Moreover, although bioluminescence confers an advantage for V. fischeri during colonization of its natural host, Euprymna scolopes, the phr mutant colonized this host to the same level as the wild type. Taken together, our results indicate that at least in V. fischeri strain ES114, the benefits of bioluminescence during symbiotic colonization are not mediated by photolyase, and although some UV resistance mechanism may be coregulated with bioluminescence, we found no evidence that light production benefits cells by stimulating photolyase in this strain.     

烟曲霉黑色素的研究进展

烟曲霉(Aspergillus fumigatus)是一种分布于世界各地的腐生真菌,属于人类临床常见的三大机会性致病真菌之一,是侵袭性曲霉菌病的主要病原菌。烟曲霉可以产生DHN-黑色素(dihydroxynaphthalene melanin)和脓黑素(pyomelanin)这2种类型黑色素。本综述介绍烟曲霉黑色素产生的遗传代谢途径、功能以及与宿主免疫系统相互作用的最新认识,有助于更好地理解烟曲霉的病理生理特征,为烟曲霉感染快速诊断技术和新型抗真菌药物的研发提供理论依据。     

Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics

Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 10⁶ to 10⁷ bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×10⁸ bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to challenge mice treated with drugs or vaccines, and test the survival, dissemination, and transmission of leptospires between environment and hosts.