Systemic fungal infections constitute a major public health problem in many parts of the world, in both developed and developing countries. The problem is increasing as a result of growing populations of immunocompromised individuals such as those with AIDS or undergoing treatment with immunosuppressive drugs. Further, the alarming emergence of infections outside traditional transplant and oncology units demonstrates that such infections are no longer restricted to the most severely immunosuppressed. The paucity of safe and effective antimycotic drugs, especially for systemic infections, compounds the problem.
Penicillium marneffei is an emerging human pathogenic fungus that causes a fatal systemic mycosis. P. marneffei is endemic to South East Asia and, as a result of its escalating incidence, now represents an “AIDS-defining pathogen” in this region. Approximately 30 percent of AIDS patients in South East Asia have P. marneffei infections. As a consequence of the increasing frequency of travel to Asia, the capacity of P. marneffei to exist in the absence of a host as a saprophyte, and the rate at which the pathogen can infect immunocompromised populations, P. marneffei is spreading throughout the world, with confirmed cases of P. marneffei infection reported in Europe, North America, Africa, and Australia. Although P. marneffei infections are detected primarily in immunocompromised patients, cases of infection in immunocompetent patients have also been reported.Untreated P. marneffei infections are fatal and, although early diagnosis and treatment can be effective, continued treatment is required to prevent relapse.
Like a number of other fungal pathogens,P. marneffei exhibits dimorphic growth and hence can grow as two distinct cellular forms: unicellular yeast and multicellular hyphae. P. marneffeiis the only known dimorphic Penicillium species, and the switch between growth forms is strictly regulated by temperature. At 25°C, in the saprophytic growth phase, P. marneffei grows as multinucleate, septate, branched hyphae. These hyphae produce conidia, the infectious agent, from specialized multicellular structures termed conidiophores in a developmental process called conidiation (asexual development). When switched to 37°C, P. marneffei undergoes a developmental process termed arthroconidiation, which results in the liberation of uninucleate yeast cells that then divide by fission. The yeast cells are the pathogenic form, and multiple yeast cells are evident in the pulmonary alveolar macrophages and peripheral blood mononuclear cells of infected individuals. The unicellular yeast cells are very similar to those of other fungal pathogens, such as Histoplasma capsulatum, Cryptococcus neoformans, and Candida albicans, except that P. marneffei yeast cells divide by fission, whereas the other pathogenic yeasts divide by budding. Thus, P. marneffei is more closely related to the monomorphic pathogen Aspergillus fumigatus than to H. capsulatum. This unique evolutionary position makes P. arneffei an excellent system in which to study the evolution of dimorphic switching and pathogenicity.
Transcriptional Control
Molecular genetic studies in my laboratory have shown that dimorphic switching and conidiation share a number of molecular controls. Conidiation in P. marneffei is controlled by the sequential activation of a central transcriptional cascade consisting of the regulatory factors BrlA and AbaA and a novel factor, WetA. BrlA is a C2H2 zinc finger protein that is repressed by a signaling cascade originating from the heterotrimeric Gα subunit GasA. GasA promotes vegetative growth over development and is activated by an unidentified mechanism. BrlA then activates abaA, which in turn feedback-activates brlA, its own expression, and that of the downstream gene wetA. Both independently and together, these factors activate the expression of different classes of morphogenetic genes. P. marneffeiabaA, but not brlA, is expressed during dimorphic switching and is required for the coupling of nuclear and cell division during yeast morphogenesis. Three pieces of evidence suggest that brlA is actively repressed during yeast growth: (1) ectopic expression of brlA in yeast or hyphal cells inhibits growth and drives conidial formation, (2) dominant negative gasA alleles drive brlA expression and conidial formation in hyphal cells but not in yeast cells, and (3) abaA feedback activates brlA expression in hyphal cells but not in yeast cells. Therefore, components of this regulatory cascade are used for conidiation and dimorphic switching, and evidence points to the active “insulation” of these two developmental pathways, so that triggering one does not inappropriately trigger the other.
Fungi, plants, and some prokaryotes, but not animals, are able to use compounds such as acetate and fatty acids as carbon sources via the glyoxylate bypass (consisting of the enzymes isocitrate lyase and malate synthase). This pathway is important for pathogenicity in a number of microorganisms. We have shown that P. marneffeiacuD (isocitrate lyase) expression is induced during the hyphal-to-yeast dimorphic switch and by acetate. Induction of acuD in yeast cells is not unexpected, given that these cells exist in the phagolysosomal system of infected macrophages in the host, where fatty acids are likely available carbon sources. Acetate-inducible expression of acuD is reduced but not abolished in yeast cells of a ΔabaA mutant, whereas temperature-dependent induction is almost completely abolished, showing that abaA is responsible for acuD expression at 37°C. In most fungi where it has been examined, the major acetate-dependent activator of isocitrate lyase genes is a conserved Zn(II)2Cys6 binuclear cluster, DNA-binding, transcriptional activator. Therefore, this factor and AbaA may control temperature- and acetate-dependent activation of acuD as well as other pathogenicity gene expression at 37°C.
Cell Polarity and Signaling Control
The Rho family of small GTPases are key regulators of morphogenesis and cytokinesis in response to extracellular and intracellular signals. The spatial and temporal activity of these small GTPases depends on their effectors—proteins that recruit them to particular locations in the cell or modulate their guanine nucleotide–bound state.
We have shown that two members of the Rho family play critical roles in morphogenesis of the P. marneffei cell type. P. marneffeicflA, a CDC42 ortholog, is required for polarization of yeast cells at 37°C, hyphal cells at 25°C, and germination of spores, whereas cflB, a Rac ortholog, is required for polarization of the conidiophore cell types and hyphal cells. The key to understanding how these two small GTPases control polarization of the different cell types lies in their effector proteins. PakA is a p21-activated kinase that, as we have shown, is required for germination of conidia at 37°C, but not at 25°C, and for correct morphogenesis of yeast cells in P. marneffei. The data suggest that PakA is an effector of CflA and define a 37°C-specific signaling pathway. How temperature triggers dimorphic switching is unknown. Identification of PakA as part of this signaling pathway now provides a means to identify new components.
We have used a microarray-based expression profiling approach to extend our understanding of the molecular basis of dimorphic switching. With this approach, we have identified numerous sets of genes involved in the various steps of this process. Gene deletion studies of these genes are beginning to unravel the determinants of dimorphic switching, and pathogenicity studies are identifying the components that are also required for the pathogenic potential of P. marneffei.
Last updated September 2008
