In Aspergillus fumigatus, the repetitive region of the
gene is one of the most frequently used loci for intraspecies typing of this human pathogenic mold. Using PCR amplification and Sanger ...sequencing of only a single marker,
typing is readily available to most laboratories and highly reproducible. Here, I evaluate the usefulness of the
marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. After resolving nomenclature conflicts from published studies and adding novel
types, the number of known types now adds up to 38. Their distribution mostly correlates with A. fumigatus population structure, and they are also meaningful for narrowly defined cases of azole resistance phenotypes. Isolates carrying the pandemic resistance allele TR
/L98H show signs of interclade crossing of strains with t02 or t04A, into the t11 clade. Furthermore, absolute differences in voriconazole MIC values between t02/t04B versus t11 TR
/L98H isolates indicate that the genetic background of resistance mutations may have a pivotal role in cross-resistance phenotypes and, thus, clinical outcome and environmental selection. Despite the general genetic similarity of isolates with identical
types, outcrossing into other clades is also observed. The
type alone, therefore, does not sufficiently discriminate genetic clades to be used as the sole marker in epidemiologic studies.
Aspergillus fumigatus is a ubiquitously distributed saprophytic mold and a leading cause of invasive aspergillosis in human hosts. Pandemic azole-resistant strains have emerged on a global scale, which are thought to be propagated through use of azole-based fungicides in agriculture. To perform epidemiologic studies, genetic typing of large cohorts is key. Here, I evaluate the usefulness of the frequently used
marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. The phylogenetic distribution of
types mostly correlates with A. fumigatus population structure and is also meaningful for narrowly defined cases of azole resistance phenotypes. Nevertheless, outcrossing of
into other clades is also observed. The
type alone, therefore, does not sufficiently discriminate genetic clades and should not be used as the sole marker in epidemiologic studies.
Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune ...responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.
Infectious diseases are worldwide a major cause of morbidity and mortality. Fast and specific detection of pathogens such as bacteria is needed to combat these diseases. Optimal methods would be ...non-invasive and without extensive sample-taking/processing. Here, we developed a set of near infrared (NIR) fluorescent nanosensors and used them for remote fingerprinting of clinically important bacteria. The nanosensors are based on single-walled carbon nanotubes (SWCNTs) that fluoresce in the NIR optical tissue transparency window, which offers ultra-low background and high tissue penetration. They are chemically tailored to detect released metabolites as well as specific virulence factors (lipopolysaccharides, siderophores, DNases, proteases) and integrated into functional hydrogel arrays with 9 different sensors. These hydrogels are exposed to clinical isolates of 6 important bacteria (Staphylococcus aureus, Escherichia coli,…) and remote (≥25 cm) NIR imaging allows to identify and distinguish bacteria. Sensors are also spectrally encoded (900 nm, 1000 nm, 1250 nm) to differentiate the two major pathogens P. aeruginosa as well as S. aureus and penetrate tissue (>5 mm). This type of multiplexing with NIR fluorescent nanosensors enables remote detection and differentiation of important pathogens and the potential for smart surfaces.
The incidence of azole resistance in
species has increased over the past years, most importantly for
. This is partially attributable to the global spread of only a few resistance alleles through the ...environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant
strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of
genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.
MALDI‐TOF MS‐based species identification has found its place in many clinical routine diagnostic laboratories over the past years. Several well‐established commercial systems exist and these allow ...precise analyses not only among bacteria, but also among clinically important yeasts. This methodology shows higher precision than biochemical and microscopic methods at significantly reduced turnaround times. Furthermore, the differentiation of different filamentous fungi including most dermatophytes and zygomycetes has been established. The direct identification of yeasts from blood culture bottles will be possible in a routine fashion with new standardized procedures. In addition to species identification, the MALDI‐TOF MS technology offers several further possibilities, like assays to detect or predict resistance phenotypes in fungi as well as subtyping approaches to detect clinically relevant subgroups. The differences between the commercial systems are discussed with respect to fungi and an overview of their performances provided. Factors influencing outcome of MALDI‐TOF‐based species identification are discussed.
Abstract
Aspergillus fumigatus is a major fungal pathogen of humans but its two closest relatives, Aspergillus fischeri and Aspergillus oerlinghausenensis, are not. Steenwyk et al. examined ...whether...
Abstract
Aspergillus fumigatus is a major human pathogen. In contrast, Aspergillus fischeri and the recently described Aspergillus oerlinghausenensis, the two species most closely related to A. fumigatus, are not known to be pathogenic. Some of the genetic determinants of virulence (or “cards of virulence”) that A. fumigatus possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. To examine whether secondary metabolism-associated cards of virulence vary between these species, we conducted extensive genomic and secondary metabolite profiling analyses of multiple A. fumigatus, one A. oerlinghausenensis, and multiple A. fischeri strains. We identified two cards of virulence (gliotoxin and fumitremorgin) shared by all three species and three cards of virulence (trypacidin, pseurotin, and fumagillin) that are variable. For example, we found that all species and strains examined biosynthesized gliotoxin, which is known to contribute to virulence, consistent with the conservation of the gliotoxin biosynthetic gene cluster (BGC) across genomes. For other secondary metabolites, such as fumitremorgin, a modulator of host biology, we found that all species produced the metabolite but that there was strain heterogeneity in its production within species. Finally, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis. A. fumigatus biosynthesized fumagillin and pseurotin, while A. oerlinghausenensis biosynthesized fumagillin and A. fischeri biosynthesized neither. These biochemical differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light onto the genetic and phenotypic changes associated with the evolution of fungal pathogenicity.
•STAT1 GOF mutants show normal JAK2-induced tyrosine phosphorylation.•Tc45-catalyzed dephosphorylation is unaffected by R274W and Q275A substitutions.•The kinetics of DNA binding is unchanged in the ...GOF mutants.•GOF mutations result in premature and prolonged STAT1 nuclear accumulation.•A model is proposed for a trimeric STAT1 complex inhibiting its own nuclear import.
Heterozygous gain-of-function (GOF) mutations in the cytokine-regulated transcription factor STAT1 (signal transducer and activator of transcription 1) lead to chronic mucocutaneous candidiasis (CMC). However, the molecular basis of these pathogenic missense mutations is largely unknown. In this study, we characterized in more detail the CMC-associated GOF substitution mutation of arginine-to-tryptophan at position 274 (R274W) and, in addition, the adjacent glutamine-to-alanine mutation at position 275 (Q275A). Both mutants displayed elevated tyrosine phosphorylation levels, prolonged nuclear accumulation, and increased transcriptional responses to interferon-γ (IFNγ) stimulation. No difference was observed between wild-type (WT) and mutant STAT1 in DNA sequence-specificity or dissociation kinetics from high-affinity DNA-binding elements known as gamma-activated sites (GAS). Furthermore, all variants exhibited similar cooperative DNA binding. Unexpectedly, in vitro dephosphorylation rates using the recombinant STAT1-inactivating Tc45 phosphatase in both the absence and presence of double-stranded GAS elements were similar in all STAT1 variants. Likewise, the rate of tyrosine phosphorylation by Janus kinase 2 (JAK2) was unaltered as compared to the WT molecule, excluding that the phenotype of these mutants is caused by either defective Tc45-catalyzed dephosphorylation or JAK2-induced hyper-activation. Interestingly, within 10 min of IFNγ exposure, the majority of R274W and Q275A molecules had entered the nucleus, whereas the wild-type protein remained predominantly cytosolic. Thus, the exchange of critical residues located at the binding interface in the antiparallel dimer conformer led to a premature accumulation of phospho-STAT1 in the nuclear compartment. In summary, our data show that the hyper-activity of the GOF mutations results, at least in part, from the premature nuclear import of the tyrosine-phosphorylated molecules and not from alterations in their phosphorylation or dephosphorylation rates.
PDF
