Alex Andrianopoulos

Control of morphogenesis and actin localization by the Penicillium marneffei RAC homolog

Rac proteins control polarized growth in many organisms but the specific function of these proteins remains undefined. In this study, we describe the cloning and functional characterization of a RAC homolog, cflB, from the dimorphic fungus Penicillium marneffei. P. marneffei produces asexual spores on complex structures (conidiophores) and switches between hyphal and yeast growth. CflB colocalizes with actin at the tips of vegetative hyphal cells and at sites of cell division. Deletion of cflB results in cell division (septation) and growth defects in both vegetative hyphal and conidiophore cell types such that cells become depolarized, exhibit inappropriate septation and the actin cytoskeleton is severely disrupted. This data suggests that Rac proteins play a crucial role in actin dependent polarized growth and division. The CDC42 ortholog in P. marneffei, cflA, controls vegetative hyphal and yeast growth polarization but does not affect asexual development. By contrast, CflB affects cellular polarization during asexual development and hyphal growth but not during yeast growth. This shows that these two GTPases have both overlapping and distinct roles during growth and development. RAC orthologs are not found in less morphologically complex eukaryotes such as Saccharomyces cerevisiae, suggesting that RAC genes might have evolved with increasing cellular complexity.

The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth.

Penicillium marneffei is the only known species of its genus that is dimorphic. At 25°C, P. marneffei exhibits true filamentous growth and undergoes asexual development producing spores borne on complex structures called conidiophores. At 37°C, P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. We have cloned a homologue of the Aspergillus nidulans abaA gene encoding an ATTS/TEA DNA‐binding domain transcriptional regulator and shown that it is involved in both these developmental programs. Targeted deletion of abaA blocks asexual development at 25°C before spore production, resulting in aberrant conidiophores with reiterated terminal cells. At 37°C, the abaA deletion strain fails to switch correctly from multinucleate filamentous to uninucleate yeast cells. Both the transitional hyphal cells, which produce the yeast cells, and the yeast cells themselves contain multiple nuclei. Expression of the abaA gene is activated during both conidiation and the hyphal–yeast switch. Interestingly, the abaA gene of the filamentous monomorphic fungus A. nidulans can complement both conidiation and dimorphic switching defects in the P. marneffei abaA mutant. In addition, ectopic overexpression of abaA results in anucleate yeast cells and multinucleate vegetative filamentous cells. These data suggest that abaA regulates cell cycle events and morphogenesis in two distinct developmental programmes.

The CDC42 Homolog of the Dimorphic Fungus Penicillium marneffei Is Required for Correct Cell Polarization during Growth but Not Development

The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffei named cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation.

Control of morphogenesis in the human fungal pathogen Penicillium marneffei

Fungal pathogens are an increasing threat to human health due to the increasing population of immunocompromised individuals and the increased incidence of treatment-derived infections. Penicillium marneffei is an emerging fungal pathogen endemic to South-east Asia, where it is AIDS defining. Like many other fungal pathogens, P. marneffei is capable of alternating between a filamentous and a yeast growth form, known as dimorphic switching, in response to environmental stimuli. P. marneffei grows in the filamentous form at 25 degrees C and in the yeast form at 37 degrees C. During filamentous growth and in response to environmental cues, P. marneffei undergoes asexual development to form complex multicellular structures from which the infectious agents, the conidia, are produced. At 37 degrees C, P. marneffei undergoes the dimorphic switching program to produce the pathogenic yeast cells. These yeast cells are found intracellularly in the mononuclear phagocyte system of the host and divide by fission, in contrast to the budding mode of division exhibited by most other fungal pathogens. In addition, P. marneffei is evolutionarily distinct from most other dimorphic fungal pathogens and is the only known Penicillium species which exhibits dimorphic growth. The unique evolutionary history of P. marneffei and the rapidly increasing incidence of infection, coupled with the presence of both complex asexual development and dimorphic switching programs in one organism, makes this system a valuable one for the study of morphogenesis and pathogenicity. Recent development of molecular genetic techniques for P. marneffei, including DNA-mediated transformation, have greatly facilitated the study of these two important morphogenetic programs, asexual development and dimorphic switching, and we are beginning to uncover important determinants which control these events. Understand these programs is providing insights into the biology of P. marneffei and its pathogenic capacity.

TupA, the Penicillium marneffei Tup1p homologue, represses both yeast and spore development

Fungal pathogenesis is frequently associated with dimorphism – morphological changes between yeast and filamentous forms. Penicillium marneffei, an opportunistic human pathogen, exhibits temperature‐dependent dimorphism, with growth at 25°C as filamentous multinucleate hyphae switching at 37°C to uninucleate yeast cells associated with intracellular pathogenesis. The filamentous hyphae also undergo asexual development generating uninucleate spores, the infectious propagules. Both processes require a switch to coupled nuclear and cell division. Homologous regulators, including Tup1p/GROUCHO‐related WD40 repeat transcription factors, control dimorphism in Candida albicans and asexual development in Aspergillus nidulans. Unlike these fungi, P. marneffei has both developmental programmes allowing examination of common and programme‐specific controls. We show that deletion of tupA, the P. marneffei TUP1 homologue, confers reduced filamentation and inappropriate yeast morphogenesis at 25°C, in stark contrast to constitutive filamentation observed when C. albicans TUP1 is deleted. Deletion of tupA also confers premature brlA‐dependent asexual development, unlike reduced asexual development in the corresponding A. nidulans rcoA deletion mutant. Furthermore, the A. nidulans rcoA deletion mutant is self‐sterile, and we show that tupA from P. marneffei, which lacks an apparent sexual cycle, complements both the asexual and sexual development phenotypes. Therefore, TupA coordinates cell fate by promoting filamentation and repressing both spore and yeast morphogenetic programmes.

A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei

Members of the APSES protein group are basic helix–loop–helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only member of its genus to display a dimorphic growth transition. At 25°C, P. marneffei grows with a filamentous morphology and produces asexual spores from multicellular con‐idiophores. At 37°C, the filamentous morphology is replaced by yeast cells that reproduce by fission. We have cloned and characterized an APSES protein‐encoding gene from P. marneffei that has a high degree of similarity to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei showed that it is required for metula and phialide formation during conidiation but is not required for dimorphic growth. This suggests that APSES proteins may control processes that require budding (formation of the metulae and phialides, pseudohyphal growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans) but not those that require fission (dimorphic growth in P. marneffei). The A. nidulansΔstuA mutant has defects in both conidiation and mating. The P. marneffei stuA gene was capable of complementing the conidiation defect but could only inefficiently complement the sexual defects of the A. nidulans mutant. This suggests that the P. marneffei gene, which comes from an asexual species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.

mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish

Macrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology.

Saccharomyces cerevisiae TEC1 is required for pseudohyphal growth

Diverse eukaryotic organisms share developmental transcription factors with homologous DNA‐binding domains. We showed that the developmental regulator AbaA, a member of the ATTS/TEA (AbaA, TEF‐1, TEC1, Scalloped/TEF‐1, TEC1, AbaA) class of transcription factors of the filamentous fungus Aspergillus nidulans, induces pseudohyphal development in the yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of AbaA, TEC1p, is required for this morphological transition. We provide evidence that TEC1p functions in co‐operation with STE12p to induce pseudohyphal development.

Cis-Regulatory Elements in the Accord Retrotransposon Result in Tissue-Specific Expression of the Drosophila melanogaster Insecticide Resistance Gene Cyp6g1

Transposable elements are a major mutation source and powerful agents of adaptive change. Some transposable element insertions in genomes increase to a high frequency because of the selective advantage the mutant phenotype provides. Cyp6g1-mediated insecticide resistance in Drosophila melanogaster is due to the upregulation of the cytochrome P450 gene Cyp6g1, leading to the resistance to a variety of insecticide classes. The upregulation of Cyp6g1 is correlated with the presence of the long terminal repeat (LTR) of an Accord retrotransposon inserted 291bp upstream of the Cyp6g1 transcription start site. This resistant allele (DDT-R) is currently at a high frequency in D. melanogaster populations around the world. Here, we characterize the spatial expression of Cyp6g1 in insecticide-resistant and -susceptible strains. We show that the Accord LTR insertion is indeed the resistance-associated mutation and demonstrate that the Accord LTR carries regulatory sequences that increase the expression of Cyp6g1 in tissues important for detoxification, the midgut, Malpighian tubules, and the fat body. This study provides a significant example of how changes in tissue-specific gene expression caused by transposable-element insertions can contribute to adaptation.

The Ras and Rho GTPases genetically interact to co-ordinately regulate cell polarity during development in Penicillium marneffei

Ras and Rho GTPases have been examined in a wide variety of eukaryotes and play varied and often overlapping roles in cell polarization and development. Studies in Saccharomyces cerevisiae and mammalian cells have defined some of the central activities of these GTPases. However, these paradigms do not explain the role of these proteins in all eukaryotes. Unlike yeast, but like more complex eukaryotes, filamentous fungi have Rac‐like proteins in addition to Ras and Cdc42. To investigate the unique functions of these proteins and determine how they interact to co‐ordinately regulate morphogenesis during growth and development we undertook a genetic analysis of GTPase function by generating double mutants of the Rho GTPases cflA and cflB and the newly isolated Ras GTPase rasA from the dimorphic pathogenic fungus, Penicillium marneffei. P. marneffei growth at 25°C is as multinucleate, septate, branched hyphae which are capable of undergoing asexual development (conidiation), while at 37°C, uninucleate pathogenic yeast cells which divide by fission are produced. Here we show that RasA (Ras) acts upstream of CflA (Cdc42) to regulate germination of spores and polarized growth of both hyphal and yeast cells, while also exhibiting CflA‐independent activities. CflA (Cdc42) and CflB (Rac) co‐ordinately control hyphal cell polarization despite also having unique roles in regulating conidial germination and polarized growth of yeast cells (CflA) and polarized growth of conidiophore cell types and hyphal branching (CflB).