Strategies for the molecular genetic manipulation and visualization of the human fungal pathogen Penicillium marneffei

Boyce, Kylie J.; Bugeja, Hayley E; Weerasinghe, Harshini; Payne, Michael; Schreider, Lena

doi:10.4148/1941-4765.1009

Cited by 15 publications

(17 citation statements)

References 25 publications

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“…Slides were fixed by soaking them in a solution of 4% para‐n‐formaldehyde in PME (50 mM PIPES pH 6.7, 1 mM MgSO 4 , 20 mM EGTA) for 30 min, followed by two 5 min PME washes and stained using fluorescent brightener 28 (Calcofluor white, CAL) or Hoechst 33258. Live staining using NBT and Fillipin was performed as previously described (Semighini and Harris, ; Boyce et al ., ). Slides were Visualization of slides using bright field (BF), differential interference contrast (DIC) or epifluorescence optics with a Reichart Jung Polyvar II microscope and captured using a SPOT CCD camera (Diagnostic Instruments), or Olympus BX60 microscope and DP71 camera.…”

Section: Methodsmentioning

confidence: 97%

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

Bugeja

Hynes

Andrianopoulos

2013

Molecular Microbiology

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SummaryFungi 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 C2H2 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.

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Section: Methodsmentioning

confidence: 97%

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

Bugeja

Hynes

Andrianopoulos

2013

Molecular Microbiology

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“…Sequences of primers are provided in Supplementary Table 2. Deletion constructs were created using the modified Gateway TM method [45]. The deletion constructs of the following three genes were created using pHW7771 containing the pDONR-pyrGBlaster cassette.…”

Section: Molecular Methods and Plasmid Constructionmentioning

confidence: 99%

A unique aspartyl protease gene expansion in Talaromyces marneffei plays a role in growth inside host phagocytes

et al. 2019

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Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes (popparalogue of pep) and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei. ARTICLE HISTORY

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“…Deletion constructs were created using a modified Gateway™ method (Boyce et al 2012). The deletion construct of msgA was created using pHW7711 containing the pDONR- pyrG cassette.…”

Section: Methodsmentioning

confidence: 99%

The novelDblhomology/BAR domain protein, MsgA, ofTalaromyces marneffeiregulates yeast morphogenesis during growth inside host cells

Weerasinghe

Bugeja

Andrianopoulos

2018

Preprint

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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.

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Strategies for the molecular genetic manipulation and visualization of the human fungal pathogen Penicillium marneffei

Cited by 15 publications

References 25 publications

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

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

A unique aspartyl protease gene expansion in Talaromyces marneffei plays a role in growth inside host phagocytes

The novelDblhomology/BAR domain protein, MsgA, ofTalaromyces marneffeiregulates yeast morphogenesis during growth inside host cells

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