CYP51 Paralogue Structure Is Associated with Intrinsic Azole Resistance in Fungi.

Azoles are the most commonly used clinical antifungal therapy and also play an important role in control of plant pathogens. Intrinsic resistance to the azole class of fungicides, which target lanosterol demethylase (CYP51), is observed in many fungal species; however, the mechanisms underpinning this phenomenon are unknown. In this study, 5 azole-resistant Penicillium isolates from patients attending the UK National Aspergillosis Centre that could not be morphologically identified to species level were analyzed by genome sequencing. The genomes and CYP51 paralogue structure from these isolates were compared with those of 46 representative fungal isolates to identify to species level and examine possible mechanisms of drug resistance. Analysis of CYP51 paralogues showed that azole-resistant isolates from this study (n = 2) and from public databases (n = 6) contained a new CYP51 paralogue, CYP51D, which was associated with azole resistance in 6/8 cases and never occurred in azole-sensitive species (46/46 tested). Furthermore, one isolate from this study and an azole-resistant Aspergillus fumigatiaffinis isolate were shown to encode a CYP51A paralogue, CYP51A2. Introduction of CYP51A2 to the closely related but azole-sensitive Aspergillus fumigatus resulted in azole resistance. The identification of novel CYP51A and CYP51D paralogues in resistant fungi and the observation that resistance to azoles can be conferred by introducing a CYP51A paralogue from a resistant species into an azole-sensitive species are a potentially important new azole resistance mechanism. IMPORTANCE Azole antifungals are the main treatment for fungal disease in humans. Many species are intrinsically resistant to azoles-in other words all members of the species are resistant without prior exposure-and we do not understand why. In this study, we serendipitously discovered that many intrinsically resistant species have alternative or extra copies of the azole target gene, CYP51. Transfer of one of these genes from a resistant species to a sensitive one resulted in drug resistance, showing that the extra copies of CYP51 can confer drug resistance. Understanding how clinically important species are resistant to therapy allows us to predict whether a species could be resistant from genome sequence.

Detection of Pneumocystis jirovecii by quantitative real-time PCR in oral rinses from Pneumocystis pneumonia asymptomatic human immunodeficiency virus patients.

Pneumocystis pneumonia (PCP) is a potentially life-threatening fungal infection usually seen in immunocompromised patients. Pneumocystis jirovecii can be easily detected from oral rinse samples in HIV patients with suspected PCP. In this study, a quantitative real-time PCR assay was used to establish the frequency of detection of P. jirovecii in oral rinses from HIV patients without respiratory symptoms or suspicion of PCP. Two saline oral rinses were collected from 100 ambulant HIV patients and from 60 COPD patients (comparator group). Four HIV patients were positive for P. jirovecii. In three patients, the first sample was positive and in one the second one was positive. One of these patients was on PCP prophylaxis and had a CD4+ count of 76 cells/mm3. The mean CD4+ count for all patients was 527 cells/mm3. All qRT-PCR test results for the COPD patients were negative. No patient developed PCP at six months follow-up. The qRT-PCR assay can be used to detect P. jirovecii DNA in oral rinse samples from HIV patients without evident clinical symptoms, however the oral carriage of this fungus was rare in our cohort of patients. In conclusion, although rare, a positive oral rinse P. jirovecii result may reflect colonisation, in particular in patients with HIV. This needs to be kept in mind when using oral rinses and qRT-PCR in the diagnosis of P. jirovecii infection.

Genetic susceptibility to severe asthma with fungal sensitization.

Severe asthma is problematic and its pathogenesis poorly understood. Fungal sensitization is common, and many patients with severe asthma with fungal sensitization (SAFS), used to denote this subgroup of asthma, respond to antifungal therapy. We have investigated 325 haplotype-tagging SNPs in 22 candidate genes previously associated with aspergillosis in patients with SAFS, with comparisons in atopic asthmatics and healthy control patients, of whom 47 SAFS, 279 healthy and 152 atopic asthmatic subjects were genotyped successfully. Significant associations with SAFS compared with atopic asthma included Toll-like receptor 3 (TLR3) (p = .009), TLR9 (p = .025), C-type lectin domain family seven member A (dectin-1) (p = .043), interleukin-10 (IL-10) (p = .0010), mannose-binding lectin (MBL2) (p = .007), CC-chemokine ligand 2 (CCL2) (2 SNPs, p = .025 and .041), CCL17 (p = .002), plasminogen (p = .049) and adenosine A2a receptor (p = .024). These associations differ from those found in ABPA in asthma, indicative of contrasting disease processes. Additional and broader genetic association studies in SAFS, combined with experimental work, are likely to contribute to our understanding of different phenotypes of problematic asthma.

Aspergillus fumigatus proteases, Asp f 5 and Asp f 13, are essential for airway inflammation and remodelling in a murine inhalation model.

BACKGROUND: In susceptible individuals, exposure to Aspergillus fumigatus can lead to the development of atopic lung diseases such as allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). Protease allergens including Asp f 5 and Asp f 13 from Aspergillus fumigatus are thought to be important for initiation and progression of allergic asthma. OBJECTIVE: To assess the importance of secreted protease allergens Asp f 5 (matrix metalloprotease) and Asp f 13 (serine protease) in Aspergillus fumigatus-induced inflammation, airway hyperactivity, atopy and airway wall remodelling in a murine model following chronic exposure to secreted allergens. METHODS: BALB/c mice were repeatedly intranasally dosed over the course of 5 weeks with culture filtrate from wild-type (WT), Asp f 5 null (∆5) or Asp f 13 null (∆13) strains of Aspergillus fumigatus. Airway hyper-reactivity was measured by non-invasive whole-body plethysmography, Th2 response and airway inflammation by ELISA and cell counts, whilst airway remodelling was assessed by histological analysis. RESULTS: Parent WT and ∆5 culture filtrates showed high protease activity, whilst protease activity in ∆13 culture filtrate was low. Chronic intranasal exposure to the three different filtrates led to comparable airway hyper-reactivity and Th2 response. However, protease allergen deleted strains, in particular ∆13 culture filtrate, induced significantly less airway inflammation and remodelling compared to WT culture filtrate. CONCLUSION: Aspergillus fumigatus-secreted allergen proteases, Asp f 5 and Asp f 13, are important for recruitment of inflammatory cells and remodelling of the airways in this murine model. However, deletion of a single allergen protease fails to alleviate airway hyper-reactivity and allergic immune response. Targeting protease activity of Aspergillus fumigatus in conditions such as SAFS or ABPA may have beneficial effects in preventing key aspects of airway pathology.

DL-2-hydroxyisocaproic acid attenuates inflammatory responses in a murine Candida albicans biofilm model.

Chronic biofilm infections are often accompanied by a chronic inflammatory response, leading to impaired healing and increased, irreversible damage to host tissues. Biofilm formation is a major virulence factor for Candida albicans and a challenge for treatment. Most current antifungals have proved ineffective in eradicating infections attributed to biofilms. The biofilm structure protects Candida species against antifungals and provides a way for them to evade host immune systems. This leads to a very distinct inflammatory response compared to that seen in planktonic infections. Previously, we showed the superior efficacy of dl-2-hydroxyisocaproic acid (HICA) against various bacteria and fungi. However, the immunomodulatory properties of HICA have not been studied. Our aim was to investigate the potential anti-inflammatory response to HICA in vivo. We hypothesized that HICA reduces the levels of immune mediators and attenuates the inflammatory response. In a murine model, a robust biofilm was formed for 5 days in a diffusion chamber implanted underneath mouse skin. The biofilm was treated for 12 h with HICA, while caspofungin and phosphate-buffered saline (PBS) were used as controls. The pathophysiology and immunoexpression in the tissues surrounding the chamber were determined by immunohistochemistry. Histopathological examination showed an attenuated inflammatory response together with reduced expression of matrix metalloproteinase 9 (MMP-9) and myeloperoxidase (MPO) compared to those of chambers containing caspofungin and PBS. Interestingly, the expression of developmental endothelial locus 1 (Del-1), an antagonist of neutrophil extravasation, increased after treatment with HICA. Considering its anti-inflammatory and antimicrobial activity, HICA may have enormous therapeutic potential in the treatment of chronic biofilm infections and inflammation, such as those seen with chronic wounds.

Reduced expression of TLR3, TLR10 and TREM1 by human macrophages in Chronic cavitary pulmonary aspergillosis, and novel associations of VEGFA, DENND1B and PLAT.

Chronic cavitary pulmonary aspergillosis (CCPA) is an uncommon but serious pulmonary disease of humans, with an annual mortality rate of 10-30%. It is caused by the fungus Aspergillus fumigatus. Patients are overtly immunocompetent; however, some immunogenetic defect is likely. To investigate this, we performed a genetic association study analysing biologically plausible candidate genes in 112 CCPA patients and 279 healthy controls, and investigated gene expression in monocyte-derived macrophages from patients and controls at baseline and during stimulation with A. fumigatus. Single-nucleotide polymorphisms (SNPs) associated with CCPA were found in TLR1, CLEC7A (dectin-1), PLAT (n=2), VEGFA, and DENND1B. Macrophages from CCPA patients showed low TLR3 and TLR10 expression and high TREM1 expression at baseline, as compared with macrophages from healthy subjects, with major expression differences being seen in most Toll-like receptors (TLRs) during 9 h of co-culture with A. fumigatus. The differences in baseline expression between the healthy and CCPA groups suggest roles for TLR3 and TLR10 in susceptibility to CCPA, and the association of SNPs in PLAT (n=2), VEGFA and DENND1B supports novel roles for plasminogen activation and angiogenesis and of these genes specifically in susceptibility to CCPA.

A prominent role for the IL1 pathway and IL15 in susceptibility to chronic cavitary pulmonary aspergillosis.

Chronic cavitary pulmonary aspergillosis (CCPA) is a progressive lung condition with a 10-30% annual mortality. Although overtly immunocompetent, some immunogenetic defect in patients is likely. To investigate a possible immunogenetic defect in CCPA, we analysed biologically plausible candidate genes in 112 CCPA patients and 279 healthy controls in a genetic association study of genes involved in the post-recognition immune response to Aspergillus fumigatus. We also compared gene expression in monocyte-derived macrophages from subjects with and without disease, both at baseline and during stimulation with A. fumigatus. Compared with macrophages from healthy subjects, CCPA macrophages showed unrestrained rises in IL1A, IL1B, IL6, IRAK2 and TRAF6 throughout the experiment, and a lack of expression of TGFB1 at 9 h. Single nucleotide polymorphisms (SNPs) associated with CCPA were found in IL1B (n = 2), IL1RN and IL15 (n = 3). Uncontrolled expression of IL1 and IL6 and continuing high levels of these cytokines may result in continuing cellular influx and pro-inflammatory responses, inhibiting disease resolution and contributing to disease progression in CCPA. The association of SNPs in IL1B, IL1RN and IL15 with CCPA supports a role for the IL1 pathway, as well as implicating the IL15 gene, in susceptibility to CCPA.

2-hydroxyisocaproic acid is fungicidal for Candida and Aspergillus species.

The amino acid derivative 2-hydroxyisocaproic acid (HICA) is a nutritional additive used to increase muscle mass. Low levels can be detected in human plasma as a result of leucine metabolism. It has broad antibacterial activity but its efficacy against pathogenic fungi is not known. The aim was to test the efficacy of HICA against Candida and Aspergillus species. Efficacy of HICA against 19 clinical and reference isolates representing five Candida and three Aspergillus species with variable azole antifungal sensitivity profiles was tested using a microdilution method. The concentrations were 18, 36 and 72 mg ml(-1) . Growth was determined spectrophotometrically for Candida isolates and by visual inspection for Aspergillus isolates, viability was tested by culture and impact on morphology by microscopy. HICA of 72 mg ml(-1) was fungicidal against all Candida and Aspergillus fumigatus and Aspergillus terreus isolates. Lower concentrations were fungistatic. Aspergillus flavus was not inhibited by HICA. HICA inhibited hyphal formation in susceptible Candida albicans and A. fumigatus isolates and affected cell wall integrity. In conclusion, HICA has broad antifungal activity against Candida and Aspergillus at concentrations relevant for topical therapy. As a fungicidal agent with broad-spectrum bactericidal activity, it may be useful in the topical treatment of multispecies superficial infections.

Fermentative 2-carbon metabolism produces carcinogenic levels of acetaldehyde in Candida albicans.

UNLABELLED: Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast acetaldehyde is a by-product of the pyruvate bypass that converts pyruvate into acetyl-Coenzyme A (CoA) during fermentation. THE AIMS OF OUR STUDY WERE: (i) to determine the levels of acetaldehyde produced by Candida albicans in the presence of glucose in low oxygen tension in vitro; (ii) to analyse the expression levels of genes involved in the pyruvate-bypass and acetaldehyde production; and (iii) to analyse whether any correlations exist between acetaldehyde levels, alcohol dehydrogenase enzyme activity or expression of the genes involved in the pyruvate-bypass. Candida albicans strains were isolated from patients with oral squamous cell carcinoma (n = 5), autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients with chronic oral candidosis (n = 5), and control patients (n = 5). The acetaldehyde and ethanol production by these isolates grown under low oxygen tension in the presence of glucose was determined, and the expression of alcohol dehydrogenase (ADH1 and ADH2), pyruvate decarboxylase (PDC11), aldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS1 and ACS2) and Adh enzyme activity were analysed. The C. albicans isolates produced high levels of acetaldehyde from glucose under low oxygen tension. The acetaldehyde levels did not correlate with the expression of ADH1, ADH2 or PDC11 but correlated with the expression of down-stream genes ALD6 and ACS1. Significant differences in the gene expressions were measured between strains isolated from different patient groups. Under low oxygen tension ALD6 and ACS1, instead of ADH1 or ADH2, appear the most reliable indicators of candidal acetaldehyde production from glucose.

Major variations in Aspergillus fumigatus arising within aspergillomas in chronic pulmonary aspergillosis.

Aspergillomas develop from progressive layers of mycelial growth on the walls of pulmonary cavities over months. Aspergillomas are characteristic of chronic pulmonary aspergillosis and are a risk factor for azole resistance. We investigated genotypic and phenotypic alterations in Aspergillus fumigatus recovered from aspergillomas. Aspergillomas were removed from three patients (two at surgery, one at autopsy) and dissected. Overall 92 colonies of A. fumigatus were isolated. Microsatellite typing was conducted to determine genetic type. Itraconazole, voriconazole and posaconazole susceptibilities were performed. The cyp51A gene was sequenced in 22 isolates. Isolates from Patient 1 (n = 25) were azole susceptible and resistant, although all cyp51A sequences were wild type, the isolates split into two distinct clades. In Patient 2, isolates were less variable (n = 10), all were azole susceptible. In Patient 3 only azole-resistant strains (n = 57) were isolated, with M220K or M220T Cyp51A alterations, and microevolution was indicated. Marked diversity was observed in isolates from these patients; revealing differences in azole susceptibility, mechanism of resistance and genetic type. Importantly, routine sampling from respiratory specimens proved suboptimal in all cases; azole resistance was missed (Patient 1), cultures were negative (Patient 2) and high-level posaconazole resistance was not detected (Patient 3).

High-level expression of cyp51B in azole-resistant clinical Aspergillus fumigatus isolates.

OBJECTIVES: Resistance to azole antifungal drugs in Aspergillus fumigatus that is not mediated by target gene mutations is now common in some locations. The aim of this study was to investigate possible new mechanisms of resistance in non-target resistance. METHODS: Twelve azole-resistant A. fumigatus isolates previously shown not to carry mutations in cyp51A were tested to determine whether the alternative cyp51B gene was overexpressed. RESULTS: Of 12 isolates one showed overinduction of cyp51B after exposure to itraconazole and another showed high constitutive expression of cyp51B. CONCLUSIONS: cyp51B overexpression is a possible azole resistance mechanism in A. fumigatus.

An improved model of the Aspergillus fumigatus CYP51A protein.

Azole resistance is an increasing clinical problem for Aspergillus fumigatus, with the majority of published resistance arising from mutations in the azole target gene CYP51A. Previous structural studies of this protein have suffered from a nonorthologous, low-homology template for homology modeling. Here we present a new model based on the human CYP51A orthologue that provides a higher-quality model for A. fumigatus CYP51A.

Aspergillus flavus: human pathogen, allergen and mycotoxin producer.

Aspergillus infections have grown in importance in the last years. However, most of the studies have focused on Aspergillus fumigatus, the most prevalent species in the genus. In certain locales and hospitals, Aspergillus flavus is more common in air than A. fumigatus, for unclear reasons. After A. fumigatus, A. flavus is the second leading cause of invasive aspergillosis and it is the most common cause of superficial infection. Experimental invasive infections in mice show A. flavus to be 100-fold more virulent than A. fumigatus in terms of inoculum required. Particularly common clinical syndromes associated with A. flavus include chronic granulomatous sinusitis, keratitis, cutaneous aspergillosis, wound infections and osteomyelitis following trauma and inoculation. Outbreaks associated with A. flavus appear to be associated with single or closely related strains, in contrast to those associated with A. fumigatus. In addition, A. flavus produces aflatoxins, the most toxic and potent hepatocarcinogenic natural compounds ever characterized. Accurate species identification within Aspergillus flavus complex remains difficult due to overlapping morphological and biochemical characteristics, and much taxonomic and population genetics work is necessary to better understand the species and related species. The flavus complex currently includes 23 species or varieties, including two sexual species, Petromyces alliaceus and P. albertensis. The genome of the highly related Aspergillus oryzae is completed and available; that of A. flavus in the final stages of annotation. Our understanding of A. flavus lags far behind that of A. fumigatus. Studies of the genomics, taxonomy, population genetics, pathogenicity, allergenicity and antifungal susceptibility of A. flavus are all required.

The link between fungi and severe asthma: a summary of the evidence.

There is current evidence to demonstrate a close association between fungal sensitisation and asthma severity. Whether such an association is causal remains to be confirmed, but this is explored by means of a detailed literature review. There is evidence from two randomised controlled trials that, in the example of allergic bronchopulmonary aspergillosis (ABPA), treatment with systemic antifungal therapy can offer a therapeutic benefit to approximately 60% of patients. ABPA is only diagnosed if a combination of clinical and immunological criteria is achieved. It is not known whether such cases are a discrete clinical entity or part of a spectrum of the pulmonary allergic response to fungi or fungal products. This paper describes the epidemiological evidence that associates severity of asthma with fungi and discusses possible pathogenetic mechanisms. Many airborne fungi are involved, including species of Alternaria, Aspergillus, Cladosporium and Penicillium, and exposure may be indoors, outdoors or both. The potential for a therapeutic role of antifungal agents for patients with severe asthma and fungal sensitisation is also explored. Not only are many patients with severe asthma desperately disabled by their disease, but, in the UK alone, asthma accounts for 1,500 deaths per yr. The healthcare costs of these patients are enormous and any treatment option merits close scrutiny. Within this report, the case for the consideration of a new term related to this association is put forward. The current authors propose the term "severe asthma with fungal sensitisation". However, it is recognised that enhanced and precise definition of fungal sensitisation will require improvements in diagnostic testing.

Relative reactivity of Aspergillus allergens used in serological tests.

Aspergillus is a common disease-causing agent, both as an allergen causing ABPA and severe asthma with fungal sensitization (SAFS) and as a pathogen causing invasive aspergillosis in immunocompromised individuals and chronic cavitating disease (CCPA) in apparently immune competent individuals. Currently detection of Aspergillus is problematic and some of the most useful tests rely on detection of antibody response to Aspergillus allergens. Here we examine the IgE antibody response to crude and recombinant allergen tests (Asp f 1, Asp f 2, Asp f 4 and Asp f 6) in individuals with allergic conditions ABPA, SAFS and in individuals with CCPA. Additionally we use recently obtained genomic information to examine the possibility of cross reaction to these allergens and show that possible cross reactive epitopes occur in several species of Aspergillus.

Recurrent embolotherapy in Dieulafoy's disease of the bronchus.

Dieulafoy's disease is a vascular anomaly characterized by the presence of a dysplastic artery that is related to an epithelial ulcer. The French surgeon Georges Dieulafoy first described it in 1898. Most frequently, it is a gastrointestinal condition, but occurrence in the bronchus has been reported in a few cases. The case of a 52-year-old man with massive hemoptysis, for which he underwent successful embolotherapy 10 years previously, is described. Over the next 10 years, he had several hospital admissions due to hemoptysis, and he underwent successful embolotherapy on each occasion. This case report underlines the importance of bronchial arteriography as the investigation of choice for massive hemoptysis.

Analyzing and correcting spectrometer temperature sensitivity.

Stability and reproducibility of the spectrometer are fundamental to the success of many modern NMR experiments. Variation in room temperature is a particularly important source of instability, in part because it can cause coherent artifacts in NMR spectra. Small changes in room temperature lead to corresponding changes in the phases, amplitudes, and frequencies of NMR signals. These can lead in turn to apparently random spectral artifacts such as t(1)-noise in two-dimensional (2D) NMR and to the incomplete cancellation of signals in difference spectra, but also, importantly, to F(1) satellite signals in 2D spectra. These "parallel diagonals" arise from the use of air conditioning, which typically forces room temperature to oscillate within a fixed band. Work to identify, quantify, and suppress sources of temperature sensitivity in a modern 300-MHz spectrometer has led to a greater than 10-fold improvement in the signal-to-artifact ratio.

Ornithine decarboxylase knockout in Tapesia yallundae abolishes infection plaque formation in vitro but does not reduce virulence toward wheat.

A knockout strain of Tapesia yallundae lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the ODC gene was isolated by polymerase chain reaction with degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced, and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the T. yallundae ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Transformation-mediated gene replacement was used to create strains that were auxotrophic for putrescine and lack ODC coding sequences. ODC knockout strains were unable to differentiate infection structures after in vitro induction and showed an abnormal hyphal branching phenotype. Pathogenicity studies on these mutants showed that, surprisingly, they are not reduced in virulence compared with nondisrupted transformants. This suggests that the strains carrying an ODC disruption can obtain sufficient polyamines from the host plant for normal growth and differentiation and, therefore, that fungal ODC may not be a suitable target for fungicides.

Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum.

Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat cell walls as the sole carbon and nitrogen source secretes numerous extracellular depolymerases, including a rapidly produced, alkaline, trypsin-like protease (SNP1). The enzyme was purified 417-fold by cation exchange chromatography and has a molecular mass of 25 kDa on sodium dodecyl sulfate gels, pI 8.7, and pH optimum of 8.5. It cleaved peptide bonds on the carboxyl side of lysine or arginine, was strongly inhibited by the trypsin inhibitors aprotinin and leupeptin and weakly by phenylmethylsulfonyl fluoride, and its activity was stimulated by calcium. SNP1 has the characteristic, conserved, fungal, trypsin N terminus. Polymerase chain reaction (PCR) primers based on this sequence and the conserved trypsin active site were used to amplify a DNA fragment that facilitated isolation of the corresponding genomic clone from a lambda library of S. nodorum. The full-length sequence confirmed its identity as a trypsin-like protease containing the N-terminal sequence of the previously purified enzyme. Infected leaf tissue contained a protease, not present in controls, that coeluted with the fungal trypsin from cation exchange, and had properties (pI and inhibitor characteristics) similar to those of the fungal trypsin. SNP1 expression in planta was detected by Northern (RNA) blotting, reverse transcription PCR, and green fluorescent protein confocal microscopy. SNP1 released hydroxyproline from wheat cell walls. The release of hydroxyproline, together with its early expression in planta, suggests that SNP1 participates in the degradation of host cell walls during infection.