Aspergillus fumigatus and aspergillosis: From basics to clinics.

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP 51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP 51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.

Aspergillus strain typing in the genomics era.

Multiple reasons may justify a need for strain typing purposes, but the most common reason is to delineate the epidemiological relationships between isolates. The availability of whole genome sequences has greatly influenced our ability to develop highly targeted and efficient strain typing methods fur these purposes. Some strain typing methods may serve dual goals: not only can they be used to discriminate between multiple isolates of a certain species, they can also aid in the recognition, identification, description and validation process of a fungal species.

Aspergillus nidulans is frequently resistant to amphotericin B.

The high failure rate of amphotericin B-based therapy in patients with Aspergillus nidulans infections may not be entirely a result of host factors as suggested previously. Innate resistance of A. nidulans to polyenes may contribute to the poor response in patients.

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essential in vivo function(s).

The single class I myosin (MYOA) of Aspergillus nidulans is essential for hyphal growth. It is generally assumed that the functions of all myosins depend on their actin-activated MgATPase activity. Here we show that MYOA mutants with no more than 1% of the actin-activated MgATPase activity of wild-type MYOA in vitro and no detectable in vitro motility activity can support fungal cell growth, albeit with a delay in germination time and a reduction in hyphal elongation. From these and other data, we conclude that the essential role(s) of myosin I in A. nidulans is probably structural, requiring little, if any, actin-activated MgATPase or motor activity, which have long been considered the defining characteristics of the myosin family.

Resistance to itraconazole in Aspergillus nidulans and Aspergillus fumigatus is conferred by extra copies of the A. nidulans P-450 14alpha-demethylase gene, pdmA.

Triazoles selectively inhibit the cytochrome P-450-dependent C-14 lanosterol alpha-demethylase (P-450 14 alpha DM), a key enzyme in ergosterol biosynthesis in fungi. To investigate mechanisms of triazole resistance in a mould, we used Aspergillus nidulans, a genetically amenable model fungus closely related to more pathogenic members of the genus. We selected for genes that would give resistance to itraconazole following transformation with a high copy genomic library of A. nidulans. In all the resistant colonies that we isolated, resistance was conferred by extra copies of the A. nidulans P-450 14 alpha DM gene, pdmA. We determined that in A. nidulans, extra copies of pdmA increase the MIC for itraconazole 36 times over wild-type controls. Similarly, transformation of an Aspergillus fumigatus strain with pITZR1 resulted in increased resistance to itraconazole. Our results indicate that triazole resistance in clinical isolates of moulds may result from amplification or overexpression of the P-450 14 alpha DM and demonstrate the utility of A. nidulans as a promising model fungus for the analysis of drug resistance and susceptibility in the pathogenic fungus A. fumigatus.

The molecular mechanisms of conidial germination.

The asexual spore, or conidium, is critical in the life cycle of many fungi because it is the primary means for dispersion and serves as a 'safe house' for the fungal genome in adverse environmental conditions. This review discusses the physiological process of germination, conidial adhesion and initiation of protein synthesis and also the regulatory pathways used to activate conidial germination. These include Ca(2+)/calmodulin-mediated signaling, the cyclic AMP/protein kinase A and the ras/mitogen-activated protein kinase pathways. Insights into the process of conidial germination will increase our understanding of the mechanisms of dormancy and sensing of environmental stimuli, and permit identification of novel therapeutic targets for the treatment of spore-borne fungal infections in plants and animals.

Conidial germination in Aspergillus nidulans requires RAS signaling and protein synthesis.

The dormant spores of Aspergillus nidulans become competent for growth and nuclear division in a process called conidial germination. To analyze the molecular details of conidial germination, we developed a genetic screen in which we identified spore germination-deficient mutants that are blocked in this process at the restrictive temperature. These mutants defined eight genes, of which we identified five. Four of the five were directly involved in translation and protein folding, and the fifth showed a high degree of homology to a malonyl CoA synthetase. These results suggest that out of a wide array of processes occurring during conidial germination, translation is essential if germination is to proceed. We also show that conidia containing a mutant-activated form of rasA, the ras homologue in A. nidulans, germinate in the absence of an inducing carbon source, suggesting an important role for rasA signaling in conidial germination. Together these data suggest a model by which a carbon source activates a ras-dependent sensory mechanism, inducing translation and leading to conidial germination. This study shows that conidial germination in A. nidulans requires protein synthesis and that the initiation of translation is linked, through an as yet to be determined signaling cascade that includes rasA, to a carbon-source-sensing apparatus.

Localization of wild type and mutant class I myosin proteins in Aspergillus nidulans using GFP-fusion proteins.

We have examined the distribution of MYOA, the class I myosin protein of the filamentous fungus Aspergillus nidulans, as a GFP fusion protein. Wild type GFP-MYOA expressed from the myoA promoter is able to rescue a conditional myoA null mutant. Growth of a strain expressing GFP-MYOA as the only class I myosin was approximately 50% that of a control strain, demonstrating that the fusion protein retains substantial myosin function. The distribution of the wild type GFP-MYOA fusion is enriched in growing hyphal tips and at sites of septum formation. In addition, we find that GFP-MYOA is also found in patches at the cell cortex. We have also investigated the effects of deletion or truncation mutations in the tail domain on MYOA localization. Mutant GFP-MYOA fusions that lacked either the C-terminal SH3 or a portion of the C-terminal proline-rich domain had subcellular distributions like wild type MYOA, consistent with their ability to complement a myoA null mutant. In contrast, mutants lacking all of the C-terminal proline-rich domain or the TH-1-like domain were mainly localized diffusely throughout the cytoplasm, but could less frequently be found in patches, and were unable to complement a myoA null mutant. The GFP-MYOA DeltaIQ mutant was localized into large bright fluorescent patches in the cytoplasm. This mutant protein was subsequently found to be insoluble.

Polarity-defective mutants of Aspergillus nidulans.

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the alpha subunit of yeast mitochondrial phenylalanyl--tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed.

Structural requirements for in vivo myosin I function in Aspergillus nidulans.

We have investigated the minimal requirements of the tail region for myosin I function in vivo using the filamentous fungus Aspergillus nidulans. The CL3 strain (McGoldrick, C. A., Gruver, C., and May, G. S. (1995) J. Cell Biol. 128, 577-587) was transformed with a variety of myoA constructs containing mutations in the IQ, TH-1-like, SH3, and proline-rich domains by frameshift or in-frame deletions of the tail domains. The resulting strains contained wild type myoA driven by the alcA promoter and a mutant myoA driven by its endogenous promoter. This strategy allowed for selective expression of the wild type and/or mutant form of MYOA by the choice of growth medium. Proper septation and hyphal branching were found to be dependent on the interaction of the IQ motifs with calmodulin, as well as, the presence of its proline-rich domain. Additionally, a single proline-rich motif was sufficient for nearly wild type MYOA function. Most surprisingly, the SH3 domain was not essential for MYOA function. These studies expand our previous knowledge of the function of MYOA to include roles in hyphal morphogenesis, septal wall formation, and cell polarity, laying the groundwork for more detailed investigations on the function of the various tail domains in MYOA.

Tyrphostin AG 494 blocks Cdk2 activation.

We have previously shown that the EGFR kinase selective tyrphostin AG 494 fails to inhibit EGFR kinase in intact cells. Yet, AG 494 proved to inhibit EGF- or serum-induced cell proliferation (Osherov et al., J. Biol. Chem. 268 (1993) 11134-11142). In this preliminary communication we show that AG 494 as well as its close analogs AG 490 and AG 555 block Cdk2 activation. In contrast, AG 1478, a more selective EGFR kinase blocker which is also active as EGFR kinase blocker in intact cells, fails to do so. AG 494 exerts its full inhibitory activity on Cdk2 activation even when added 20 h subsequent to EGF addition when Cdk2 activation is maximal. The inhibitory activity on Cdk2 activation parallels its DNA synthesis inhibitory activity, strongly suggesting that its target is one of the molecular mechanisms involved in Cdk2 activation. AG 494 and its analogs may become useful lead compounds for the development of drugs aimed at the cell cycle machinery.

Precocious sporulation and developmental lethality in yelA null mutants of Dictyostelium.

A novel developmental gene, yelA, has been found that plays as essential role in regulating terminal differentiation of Dictyostelium discoideum. Strains in which yelA is disrupted by plasmid insertion are arrested at the tight mound stage but accumulate the bright yellow pigment characteristic of mature sori. Although these mutant strains do not form fruiting bodies, many of the cells encapsulate within the mounds. Sporulation occurs about 6 hours earlier in yelA- cells than in wild-type cells, accompanied by precocious expression of a prespore gene, spiA. However, the spores are defective and lose viability over a period of several hours. Unencapsulated cells also die unless they are dissociated from the mounds and shaken in suspension. The yelA gene was isolated by plasmid rescue and found to encode a protein of 102 kDa in which the N-terminal sequence shows significant similarity to domains found in the eIF-4G subunits of the translational initiation complex eIF-4F. In wild-type cells yelA mRNA first accumulates at 8 hours of development and is maintained in both prespore and prestalk cells until culmination when it is found only is stalk cells. Mutations in yelA can partially suppress the block to sporulation in mutant strains in which either of the prestalk genes tagB or tagC is disrupted such that an encapsulation signal is not produced. It appears that premature encapsulation is normally inhibited by YelA until a signal is received from prestalk cells during culmination.

Tyrphostins. 6. Dimeric benzylidenemalononitrile tyrophostins: potent inhibitors of EGF receptor tyrosine kinase in vitro.

Benzylidenemalononitrile (BMN) tyrphostins were previously found to be potent inhibitors of EGF receptor (EGFR) tyrosine kinase activity. Since these compounds were found to compete for the substrate and sometimes with the ATP site and since EGFR acts as a dimer, we prepared a series of dimeric tyrphostins. These dimeric tyrphostins were built from two BMN units linked by various spacers and designed to fit the dimeric cross-autophosphorylation signal transduction intermediate of the EGFR tyrosine kinases. Structure-activity relationship of these potent dimeric EGF receptor tyrosine kinase inhibitors is reported.

The Dictyostelium dual-specificity kinase splA is essential for spore differentiation.

We have studied the structure and function of the Dictyostelium kinase splA. A truncated form of the splA protein exhibited primarily tyrosine kinase activity in vitro; however, it also autophosphorylated on serine and threonine residues. The kinase domain of splA exhibits approximately 38% identity to the CTR1 kinase of Arabidopsis, which is a member of the Raf family. Outside its kinase domain, splA shares homology with the byr2 kinase of S. pombe. By aligning the sequences of splA, byr2 and STE11, a homologue of byr2 in S. cerevisiae, we have identified a conserved motif that is also found in members of the Eph family of growth factor receptor tyrosine kinases. SplA is expressed throughout development with a peak during the mound stage of morphogenesis. Strains in which the splA gene had been disrupted completed fruiting body formation; however, spore cells spontaneously lysed before completing their differentiation. Northern analysis revealed the expression of the prespore marker cotB and the prestalk markers ecmA and ecmB in the mutant strain during development. The spore differentiation marker spiA was detected in the mutant spores both by northern and immunoblotting, but these cells failed to assemble spore coats. Immunoblot analysis of the developmental pattern of tyrosine phosphorylation revealed a protein that was phosphorylated in mutants but was not phosphorylated in the wild-type cells. SplA is a novel dual specificity kinase that regulates the differentiation of spore cells.

Epidermal-growth-factor-dependent activation of the src-family kinases.

The precise role of src-type kinases as signal transducers has been under intensive investigation but only in a few instances has their role been revealed in any detail. Thus, src, fyn and yes are activated upon stimulation by platelet-derived growth factor or colony-stimulating factor in cells expressing high levels of these receptors. Activation of src-family kinases by other receptor tyrosine kinases such as the epidermal-growth-factor (EGF) receptor has not been directly demonstrated. In this report, we demonstrate EGF-dependent activation of src-family tyrosine kinases in NIH3T3 cells overexpressing the human EGF receptor. Activation is rapid (< 1 min) and persistent (up to 16 h). Furthermore, we show a correlation between the level of EGF receptor expressed and the degree of src-family kinase activation. We show that src-family kinase activity is also activated by addition of EGF to PC12 cells, which endogenously express relatively high levels of EGF receptor. Most strikingly, we show that A431 cells, which endogenously express very high levels of EGF receptor, show 10-fold elevated src-family kinase activity as compared to DHER14 cells, and that this activity is constitutive. This activity is completely blocked by AG1478, a specific inhibitor of the EGF-receptor tyrosine kinase activity, pointing to a direct link between overexpression of the EGF receptor and enhanced src-family kinase activity. Our findings suggest that EGF-dependent src-family kinase activity is detectable only when the levels of EGF receptor reach a specific level. Additionally, high levels of EGF receptor, as in A431 cells, may contribute to the elevated activation of src-family kinases. Sustained src-family kinase activation, similar to that seen in v-src-transformed cells, may play a role in tumorogenesis and tumor maintenance.

Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors.

Septic shock results from excessive stimulation of the host immune system, especially macrophages, by lipopolysaccharide (LPS), or endotoxin, which resides on the outer membrane of bacteria. Protein tyrosine kinase inhibitors of the tyrphostin AG 126 family protect mice against LPS-induced lethal toxicity. The protection correlates with the ability of these agents to block LPS-induced production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in macrophages as well as LPS-induced production of TNF-alpha in vivo. Furthermore, this inhibitory effect correlated with the potency of AG 126 to block LPS-induced tyrosine phosphorylation of a p42MAPK protein substrate in the murine macrophage.

Tyrphostins. 3. Structure-activity relationship studies of alpha-substituted benzylidenemalononitrile 5-S-aryltyrphostins.

In this study we describe an extension of our previous studies on cis-benzylidenemalononitrile tyrphostins. We have introduced S-aryl substituents in the 5 position (meta vis-a-vis the malononitrile moiety). We find that these compounds are potent blockers of EGFR kinase and its homolog HER-2 kinase. Interestingly, we find that certain S-aryltryphostins discriminate between EGFR and HER-2 kinase in favor of the HER-2 kinase domain by almost 2 orders of magnitude. When examined in intact cells it was found that these selective S-aryltrphostins are equipotent in inhibiting EGF dependent proliferation of NIH 3T3 harboring either the EGF receptor or the chimera EGF/neu (HER1-2). These findings suggest that the antiproliferative activity of these tyrphostins is mainly due to the inhibition of a mitogenic signaling element downstream to the growth receptor kinase.

Non-amine based analogues of lavendustin A as protein-tyrosine kinase inhibitors.

The fermentation product lavendustin A (1) is a protein-tyrosine kinase (PTK) inhibitor whose active pharmacophore has previously been shown to reside in the more simplified salicyl-containing benzylamine 2. Amine 2 bears some structural resemblance to two other natural product PTK inhibitors, erbstatin (3) and piceatannol (4). Non-amine containing analogues of 2 were therefore synthesized which incorporated additional aspects of either erbstatin or piceatannol. Examination of these inhibitors in immunoprecipitated p56lck, epidermal growth factor receptor (EGFR), and c-erb B-2/HER 2/neu PTK preparations showed that compound 12 (IC50 = 60 nM) was one of the most potent p56lck inhibitors reported to date. These results demonstrate that nitrogen is not an essential component of the lavendustin A pharmacophore 2 and that 1,2-diarylethanes and -ethenes bearing a salicyl moiety appear to be valuable structural motifs for the construction of extremely potent PTK inhibitors.

Selective inhibition of the epidermal growth factor and HER2/neu receptors by tyrphostins.

The HER2 (neu/erb-B2) proto-oncogene codes for a transmembrane receptor with tyrosine kinase activity and with high homology to the EGF receptor (HER1). The high incidence of HER2 overexpression in breast and ovary carcinomas prompted us to synthesize protein tyrosine kinase inhibitors (tyrphostins) which selectively inhibit the HER2 kinase activity. Two groups of tyrphostins were developed: one highly selective in inhibiting HER1 as opposed to HER2, the other highly selective in inhibiting HER2. Both the HER1 and the HER2 selective blockers were competitive with ATP binding. This suggests that even though the kinase domains of the respective receptors show an 80% degree of homology it is possible to design small molecules capable of discriminating between them. These results also show that the two kinases differ in their ATP binding sites. Mitogenic signaling induced by EGF in NIH3T3 cells overexpressing either HER1 or HER1-2 (possessing the HER2 kinase domain) was blocked identically by the agents that discriminate between the two in vitro. This paradox was further explored and elucidated. We propose that high intracellular ATP levels prevent inhibitor binding to the receptor. The antiproliferative action of the two distinct selective tyrphostins observed may result from the inhibition of a downstream element, presumably a tyrosine kinase, which mediates mitogenic signaling.