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Microbial cells sense and respond to their environment using their surface constituents. Therefore, understanding the assembly and biophysical properties of cell surface molecules is an important research topic. With its ability to observe living microbial cells at nanometer resolution and to manipulate single-cell surface molecules, atomic force microscopy (AFM) has emerged as a powerful tool in microbiology. Here, we survey major breakthroughs made in cell surface microbiology using AFM techniques, emphasizing the most recent structural and functional insights., (Copyright © 2014 Dufrêne.)

Here we show that expression of a constitutively activated RasA allele, as the sole source of Ras activity, revealed novel Ras-induced phenotypes, including excessive vacuolar expansion and spontaneous lysis of hyphal compartments. These findings highlight the requirement for balanced Ras activity in the establishment and maintenance of polarized growth in filamentous fungi.

Sexual reproduction of the human pathogen Aspergillus fumigatus (teleomorph: Neosartorya fumigata) was assumed to be absent or cryptic until recently, when fertile crosses among geographically restricted environmental isolates were described. Here, we provide evidence for mating, fruiting body development, and ascosporogenesis accompanied by genetic recombination between unrelated, clinical isolates of A. fumigatus, and this evidence demonstrates the generality and reproducibility of this long-time-undisclosed phase in the life cycle of this heterothallic fungus. Successful mating requires the presence of both mating-type idiomorphs MAT1-1 and MAT1-2, as does expression of genes encoding factors presumably involved in this process. Moreover, analysis of an A. fumigatus mutant deleted for the nsdD gene suggests a role of this conserved regulator of cleistothecium development in hyphal fusion and hence heterokaryon formation.

Genes that are essential for viability represent potential targets for the development of anti-infective agents. However, relatively few have been determined in the filamentous fungal pathogen Aspergillus fumigatus. A novel solution employing parasexual genetics coupled with transposon mutagenesis using the Fusarium oxysporum transposon impala had previously enabled the identification of 20 essential genes from A. fumigatus; however, further use of this system required a better understanding of the mode of action of the transposon itself. Examination of a range of conditions indicated that impala is activated by prolonged exposure to low temperatures. This newly identified property was then harnessed to identify 96 loci that are critical for viability in A. fumigatus, including genes required for RNA metabolism, organelle organization, protein transport, ribosome biogenesis, and transcription, as well as a number of noncoding RNAs. A number of these genes represent potential targets for much-needed novel antifungal drugs.

Proper regulation of the cyclic AMP-dependent protein kinase (PKA) pathway is required for normal growth and development in many fungi. We have reported that deletion of the PKA regulatory subunit gene, pkaR, in Aspergillus fumigatus leads to defects in germination and a hypersensitivity of conidia to oxidative stress. In this study, we further analyzed the defects of DeltapkaR conidia and found that a large proportion were abnormally larger than wild type. Because swelling and increased susceptibility to oxidative stress are characteristic of germinating conidia, we analyzed the metabolic activity of the conidia by mitochondrial staining. Whereas it required 4 h in rich medium for wild-type mitochondria to become active, DeltapkaR conidia harbored active mitochondria in the absence of a germinant. Furthermore, conidia of the mutant showed a dramatic loss in viability upon short-term storage in water, indicating starvation-induced death. Taken together, our data suggest that PKA activity regulates metabolic activation of resting conidia. Additionally, the DeltapkaR mutant displayed an abnormal abundance of hyphal nuclei and had increased transcript levels of several cell cycle regulatory genes. These data indicate an important role for PKA in the nuclear duplication cycle of A. fumigatus.

Conidiation (asexual sporulation) is a key developmental process in filamentous fungi. We examined the gene regulatory roles of the Aspergillus fumigatus developmental transcription factors StuAp and BrlAp during conidiation. Conidiation was completely abrogated in an A. fumigatus DeltabrlA mutant and was severely impaired in a DeltastuA mutant. We determined the full genome conidiation transcriptomes of wild-type and DeltabrlA and DeltastuA mutant A. fumigatus and found that BrlAp and StuAp governed overlapping but distinct transcriptional programs. Six secondary metabolite biosynthetic clusters were found to be regulated by StuAp, while only one cluster exhibited BrlAp-dependent expression. The DeltabrlA mutant, but not the DeltastuA mutant, had impaired downregulation of genes encoding ribosomal proteins under nitrogen-limiting, but not carbon-limiting, conditions. Interestingly, inhibition of the target of rapamycin (TOR) pathway also caused downregulation of ribosomal protein genes in both the wild-type strain and the DeltabrlA mutant. Downregulation of these genes by TOR inhibition was associated with conidiation in the wild-type strain but not in the DeltabrlA mutant. Therefore, BrlAp-mediated repression of ribosomal protein gene expression is not downstream of the TOR pathway. Furthermore, inhibition of ribosomal protein gene expression is not sufficient to induce conidiation in the absence of BrlAp.

The calcineurin pathway is a critical signal transduction pathway in fungi that mediates growth, morphology, stress responses, and pathogenicity. The importance of the calcineurin pathway in fungal physiology creates an opportunity for the development of new antifungal therapies that target this critical signaling pathway. In this study, we examined the role of the zinc finger transcription factor Crz1 homolog (CrzA) in the physiology and pathogenicity of the opportunistic human fungal pathogen Aspergillus fumigatus. Genetic replacement of the crzA locus in A. fumigatus resulted in a strain with significant defects in conidial germination, polarized hyphal growth, cell wall structure, and asexual development that are similar to but with differences from defects seen in the A. fumigatus DeltacnaA (calcineurin A) strain. Like the DeltacnaA strain, the DeltacrzA strain was incapable of causing disease in an experimental persistently neutropenic inhalational murine model of invasive pulmonary aspergillosis. Our results suggest that CrzA is an important downstream effector of calcineurin that controls morphology in A. fumigatus, but additional downstream effectors that mediate calcineurin signal transduction are likely present in this opportunistic fungal pathogen. In addition, the importance of CrzA to the production of disease is critical, and thus CrzA is an attractive fungus-specific antifungal target for the treatment of invasive aspergillosis.

The alb1 (pksP) gene has been reported as a virulence factor controlling the pigmentation and morphology of conidia in Aspergillus fumigatus. A recent report suggested that laeA regulates alb1 expression and conidial morphology but not pigmentation in the A. fumigatus strain AF293. laeA has also been reported to regulate the synthesis of secondary metabolites, such as gliotoxin. We compared the role of laeA in the regulation of conidial morphology and the expression of alb1 and gliP in strains B-5233 and AF293, which differ in colony morphology and nutritional requirements. Deletion of laeA did not affect conidial morphology or pigmentation in these strains, suggesting that laeA is not involved in alb1 regulation during conidial morphogenesis. Deletion of laeA, however, caused down-regulation of alb1 during mycelial growth in a liquid medium. Transcription of gliP, involved in the synthesis of gliotoxin, was drastically reduced in B-5233laeADelta, and the gliotoxin level found in the culture filtrates was 20% of wild-type concentrations. While up-regulation of gliP in AF293 was comparable to that in B-5233, the relative mRNA level in AF293laeADelta was about fourfold lower than that in B-5233laeADelta. Strain B-5233laeADelta caused slower onset of fatal infection in mice relative to that with B-5233. Histopathology of sections from lungs of infected mice corroborated the survival data. Culture filtrates from B-5233laeADelta caused reduced death in thymoma cells and were less inhibitory to a respiratory burst of neutrophils than culture filtrates from B-5233. Our results suggest that while laeA is not involved in the regulation of alb1 function in conidial morphology, it regulates the synthesis of gliotoxin and the virulence of A. fumigatus.

The opportunistic human pathogen Aspergillus fumigatus produces a large quantity of asexual spores (conidia), which are the primary agent causing invasive aspergillosis in immunocompromised patients. We investigated the mechanisms controlling asexual sporulation (conidiation) in A. fumigatus via examining functions of four key regulators, GpaA (Galpha), AfFlbA (RGS), AfFluG, and AfBrlA, previously studied in Aspergillus nidulans. Expression analyses of gpaA, AfflbA, AffluG, AfbrlA, and AfwetA throughout the life cycle of A. fumigatus revealed that, while transcripts of AfflbA and AffluG accumulate constantly, the latter two downstream developmental regulators are specifically expressed during conidiation. Both loss-of-function AfflbA and dominant activating GpaA(Q204L) mutations resulted in reduced conidiation with increased hyphal proliferation, indicating that GpaA signaling activates vegetative growth while inhibiting conidiation. As GpaA is the primary target of AfFlbA, the dominant interfering GpaA(G203R) mutation suppressed reduced conidiation caused by loss of AfflbA function. These results corroborate the hypothesis that functions of G proteins and RGSs are conserved in aspergilli. We then examined functions of the two major developmental activators AfFluG and AfBrlA. While deletion of AfbrlA eliminated conidiation completely, null mutation of AffluG did not cause severe alterations in A. fumigatus sporulation in air-exposed culture, implying that, whereas the two aspergilli may have a common key downstream developmental activator, upstream mechanisms activating brlA may be distinct. Finally, both AffluG and AfflbA mutants showed reduced conidiation and delayed expression of AfbrlA in synchronized developmental induction, indicating that these upstream regulators contribute to the proper progression of conidiation.

Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The delta cnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the delta cnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the delta cnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the delta cnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

The akuA gene encoding the Ku70 component of the nonhomologous end-joining machinery was deleted in the opportunistic pathogen Aspergillus fumigatus. No obvious phenotype could be assessed for the corresponding mutant strain but relative frequencies of homologous recombination were increased as deduced from targeting the laccase-encoding abr2 gene.

To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuB(KU80)). Homologous integration reached about 80% for both calcineurin A (calA) and polyketide synthase pksP (alb1) genes in the akuB(KU80) mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuB(KU80) had no influence on pathogenicity in a low-dose murine infection model.

The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The DeltarasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited approximately 65% survival compared to approximately 10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.

When the essential and distinctive cell walls of either pathogenic or nonpathogenic fungi break, cytoplasmic membranes rupture and fungi die. This fungicidal activity was discovered previously on nonproliferating Saccharomyces cerevisiae cells treated briefly with the oxidative tool and anticancer drug family of bleomycins. The present studies investigated effects of bleomycin on growing fungal organisms. These included the medically important Aspergillus fumigatus and Cryptococcus neoformans, as well as the emerging human pathogen and fungal model, S. cerevisiae. Bleomycin had its highest potency against A. fumigatus. Scanning electron microscopy and thin-section transmission electron microscopy were used to study morphological growth characteristics. Killing and growth inhibition were also measured. Long, thin, and segmented hyphae were observed when A. fumigatus was grown without bleomycin but were never observed when the mold was grown with the drug. Bleomycin arrested conidial germination, hyphal development, and the progression and completion of cell wall septation. Similarly, the drug inhibited the construction of yeast cell wall septa, preventing cytokinesis and progression in the cell division cycle of S. cerevisiae. Even when cytoplasms of mother and daughter cells separated, septation and cell division did not necessarily occur. Bizarre cell configurations, abnormally thickened cell walls at mother-daughter necks, abnormal polarized growth, large undivided cells, fragmented cells, and empty cell ghosts were also produced. This is the first report of a fungicidal agent that arrests fungal growth and development, septum formation, and cytokinesis and that also preferentially localizes to cell walls and alters isolated cell walls as well as intact cell walls on nongrowing cells.

Sixty-one isolates and collection strains of Aspergillus fumigatus were compared for their phenotypic (morphological features and isoenzyme profiles) and genotypic (restriction enzyme-generated mitochondrial DNA and ribosomal DNA profiles and random amplified polymorphic DNA patterns) features. The examined strains exhibited highly variable colony morphologies and growth rates at different temperatures, but their micromorphologies and conidial diameters were characteristic of the species. Of the isoenzymes studied, the beta-arylesterase and phosphatase patterns were the most divergent, and the 61 strains could be classified into seven groups. The glucose 6-phosphate dehydrogenase and catalase isoenzyme patterns displayed only a limited variability, while the profiles of superoxide dismutase, lactate dehydrogenase, and glutamate dehydrogenase were highly conserved. The HaeIII-generated mitochondrial DNA patterns and SmaI-digested repetitive DNA and ribosomal DNA hybridization patterns of almost all strains were also invariable. The level of variation was much higher when random amplified polymorphic DNA analysis was applied. Although the patterns of the strains were very similar with most of the primers, the application of some primers made it possible to cluster the A. fumigatus isolates into several groups. The results indicate that the random amplified polymorphic DNA technique could be used more efficiently than isoenzyme analysis for typing A. fumigatus isolates. A good correlation was found between the dendrograms obtained from the isoenzyme and random amplified polymorphic DNA data, but the isoenzyme and amplified DNA patterns did not correlate with the pathogenicity, pigment production, or geographical origin of the strains. One "A. fumigatus" strain (strain FRR 1266) exhibited unique isoenzyme, mitochondrial DNA, ribosomal DNA, and random amplified polymorphic DNA patterns; it is proposed that this strain represents a new species of the section Fumigati.

Invasive aspergillosis is a serious fungal infection caused by the proliferation and invasion of Aspergillus hyphae in tissue. Neutrophils (PMNs) are the most important line of defense against Aspergillus hyphae. To investigate the role of granulocyte colony-stimulating factor (G-CSF) and gamma interferon (IFN-gamma) against Aspergillus fumigatus, we studied the effects of the two cytokines on the oxidative burst and the capacity of normal human PMNs to damage hyphae of the organism. G-CSF enhanced PMN oxidative burst measured as superoxide anion (O2-) production in response to N-formylmethionyl leucyl phenylalanine, serum opsonized hyphae, and nonopsonized hyphae by 75, 37, and 24%, respectively, compared with control PMNs (P < 0.015). IFN-gamma also induced increases of 52, 71, and 96%, respectively, in response to the same stimuli (P < 0.006). In addition, the capacity of PMNs to damage hyphae as measured by the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MMT) colorimetric metabolic assay was significantly enhanced by G-CSF and IFN-gamma (P < 0.01 and < 0.05, respectively). The enhancement was achieved irrespective of serum opsonization of the hyphae, suggesting upregulatory actions of the two cytokines on signal pathways specific for opsonized and nonopsonized hyphae. The combination of the two cytokines exhibited an additive effect at the higher concentrations compared with the effects of the cytokines alone (P < 0.05). Pretreatment of PMNs with protein synthesis inhibitors showed that IFN-gamma activates PMN function through transcriptional regulation, whereas the effect of G-CSF does not require new proteins. These in vitro effects suggest modulatory roles for G-CSF and IFN-gamma in the host defense against Aspergillus hyphae irrespective of serum opsonization and a potential utility of the cytokines as adjuncts for the prevention and possible treatment of invasive aspergillosis.

Complement activation by Aspergillus fumigatus may play a crucial role in stimulating binding and killing of this organism by phagocytes. We examined the amount and type of C3 deposited on resting conidia, swollen conidia, and hyphae of A. fumigatus after incubation in pooled human serum. All three life forms of A. fumigatus were potent activators of the complement cascade, with deposition on the organisms of similar amounts of C3 per unit of surface area. The rate of deposition was slowest for resting conidia, although maximal deposition was still achieved within 40 min. The roles of the alternative and classical pathways were assessed by use of serum chelated with magnesium EGTA [magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and with an alternative pathway reconstituted from the six purified alternative-pathway proteins. Complement activation by resting conidia was mediated by the alternative pathway. In contrast, there was a progressive dependence on the classical pathway as the fungal particles matured into swollen conidia and then hyphae. Treatment with hydroxylamine, which disrupts ester linkages, removed 89 to 95% of the C3 bound to all three forms of A. fumigatus. This released C3 contained a mixture of C3b and iC3b, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These data demonstrate that although all three forms of A. fumigatus are potent activators of the complement system, the transition from resting conidia to swollen conidia to hyphae results in progressive changes in the manner in which the fungal particles interact with the complement system. The lack of participation of the classical pathway in complement activation by resting conidia may have important implications regarding their ability to effectively stimulate phagocytes.