The novel Dbl homology/BAR domain protein, MsgA, of Talaromyces marneffei regulates yeast morphogenesis during growth inside host cells.

Microbial pathogens have evolved many strategies to evade recognition by the host immune system, including the use of phagocytic cells as a niche within which to proliferate. Dimorphic pathogenic fungi employ an induced morphogenetic transition, switching from multicellular hyphae to unicellular yeast that are more compatible with intracellular growth. A switch to mammalian host body temperature (37 °C) is a key trigger for the dimorphic switch. This study describes a novel gene, msgA, from the dimorphic fungal pathogen Talaromyces marneffei that controls cell morphology in response to host cues rather than temperature. The msgA gene is upregulated during murine macrophage infection, and deletion results in aberrant yeast morphology solely during growth inside macrophages. MsgA contains a Dbl homology domain, and a Bin, Amphiphysin, Rvs (BAR) domain instead of a Plekstrin homology domain typically associated with guanine nucleotide exchange factors (GEFs). The BAR domain is crucial in maintaining yeast morphology and cellular localisation during infection. The data suggests that MsgA does not act as a canonical GEF during macrophage infection and identifies a temperature independent pathway in T. marneffei that controls intracellular yeast morphogenesis.

HgrA is necessary and sufficient to drive hyphal growth in the dimorphic pathogen Penicillium marneffei.

Fungi produce multiple morphological forms as part of developmental programs or in response to changing, often stressful, environmental conditions. An opportunistic pathogen of humans, Penicillium marneffei displays multicellular hyphal growth and asexual development (conidiation) in the environment at 25°C and unicellular yeast growth in macrophages at 37°C. We characterized the transcription factor, hgrA, which contains a C(2)H(2) DNA binding domain closely related to that of the stress-response regulators Msn2/4 of Saccharomyces cerevisiae. Northern hybridization analysis demonstrated that hgrA expression is specific to hyphal growth, and its constitutive overexpression prevents conidiation and yeast growth, even in the presence of inductive cues, and causes apical hyperbranching during hyphal growth. Consistent with its expression pattern, deletion of hgrA causes defects in hyphal morphogenesis and the dimorphic transition from yeast cells to hyphae. Specifically, loss of HgrA causes cell wall defects, reduced expression of cell wall biosynthetic enzymes and increased sensitvity to cell wall, oxidative, but not osmotic stress agents. These data suggest that HgrA does not have a direct role in the response to stress but is an inducer of the hyphal growth program and its activity must be downregulated to allow alternative developmental programs, including the morphogenesis of yeast cells in macrophages.

Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei.

Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway™ system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.

AreA controls nitrogen source utilisation during both growth programs of the dimorphic fungus Penicillium marneffei.

The opportunistic pathogen Penicillium marneffei displays a temperature-dependent dimorphic switching program with saprophytic hyphal growth at 25 °C and yeast growth at 37 °C. The areA gene of P. marneffei has been isolated and found to be required for the utilisation of nonpreferred nitrogen sources during both growth programs of P. marneffei, albeit to differing degrees. Based on this functional characterisation and high degree of sequence conservation with other fungal GATA factors, P. marneffei areA represents an orthologue of Aspergillus nidulans areA and Neurospora crassa NIT2. Based on this study it is proposed that AreA is likely to contribute to the pathogenicity of P. marneffei by facilitating growth in the host environment and regulating the expression of potential virulence factors such as extracellular proteases.

The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei.

Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25 degrees C and unicellular yeast growth at 37 degrees C. P. marneffei also undergoes asexual development into differentiated multicellular conidiophores bearing uninucleate spores. These morphogenetic processes require regulated changes in cell polarity establishment, cell cycle dynamics, and nuclear migration. The RFX (regulatory factor X) proteins are a family of transcriptional regulators in eukaryotes. We sought to determine how the sole P. marneffei RFX protein, RfxA, contributes to the regulation of morphogenesis. Attempts to generate a haploid rfxA deletion strain were unsuccessful, but we did isolate an rfxA(+)/rfxADelta heterozygous diploid strain. The role of RfxA was assessed using conditional overexpression, RNA interference (RNAi), and the production of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses during growth at 25 degrees C and 37 degrees C. This was also observed for the heterozygous diploid strain during growth at 37 degrees C. In contrast, overexpression of rfxA caused growth arrest during conidial germination. The data show that rfxA must be precisely regulated for appropriate nuclear division and to maintain genome integrity. Perturbations in rfxA expression also caused defects in cellular proliferation and differentiation. The data suggest a role for RfxA in linking cellular division with morphogenesis, particularly during conidiation and yeast growth, where the uninucleate state of these cell types necessitates coupling of nuclear and cellular division tighter than that observed during multinucleate hyphal growth.