Phase transition of a dimorphic fungus
penicillium marnefei
Milena KORDALEWSKA, Dorota DRAPAŁA∗
Keywords: dimorphism; Penicillium fungi
Abstract: Penicillium marneffei is a pathogenic fungus that
afflicts immune compromised individuals who live or travel in
Southeast Asia. This species is unique, because it is the only
dimorphic member of the genus Penicillium. Dimorphism results from a process, termed phase transition, which is regulated
by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37◦ C), a uninucleate yeast form develops that reproduces
by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. The dimorphic nature of P. marneffei has
attracted investigators interested in not only understanding the
underlying molecular mechanisms of its unique mode of cellular
development, but also inthe identification of novel targets that
might be exploited in the development of new chemotherapeutic
modalities.
1. Introduction
The members of the genus Penicillium are filamentous fungi (one exception is thermally dimorphic Penicillium marneffei ). Penicillium spp. are ubiquitous fungi - they
are found in soil, decaying vegetation and in the air. P. marneffei is endemic specifically in Southeast Asia (Thailand and adjacent countries, Taiwan, and India) where it
infects bamboo rats which serve as epidemiological markers and reservoirs for human
infections. Penicilliumspp. other than P. marneffei are commonly considered to be
contaminants but may cause infections, particularly in immunocompromised hosts.
P. marneffei is pathogenic particularly in patients with AIDS and its isolation from
blood is regardedas an HIV marker in endemic areas (WHO, 2007). P. marneffei infections have also been reported in non-AIDS patients with haematological malignancies and those receiving immunosuppressive therapy (Wong et al., 2001). P. marneffei
infection is acquired via inhalation and results in initial pulmonary infection, followed
by fungaemia and dissemination of the infection. The lymphatic system, liver, spleen
and bones are usually involved. Acne-like skin papules on the face, trunk, and extremities are observed during the course of the disease. The infections are often fatal
(Cheng et al., 1998; Garbino et al., 2001; Rimek, 1999).
∗ Microbiology
Department, Faculty of Chemistry, Gdańsk University of Technology
38
PhD Interdisciplinary Journal
Penicillium marneffei is the only known Penicilliumspecies that exhibits temperaturedependent dimorphic growth. At 25◦ C, it grows in a mycelial form with vegetative
conidia. Once conidia are inhaled, they undergo dimorphic switching to produce
yeast cells at 37◦ C, the core temperature of humans. Thus, pathogenicity appears to
be intimately linked to dimorphic transition. But the mechanismthat regulates this
switching remains a mystery (Chandler et al., 2008; Kummasook et al., 2011; Liu
et al., 2007).
Fig. 1. Thermal dimorphism of P. marneffei A) The mould phase of P. marneffei (slide culture incubated at 25◦ C. B) The yeast phase of P. marneffei (produced from conidia incubated for 96 hours at 37◦ C) (Chandler et al., 2008)
The dimorphic nature of P. marneffei has attracted investigators interested in
not only understanding the underlying molecular mechanisms of its unique mode
of cellular development, but also in the identification of novel targets that might be
exploited in the development of new chemotherapeutic modalities. Initial studies were
limited to the morphological features of clinical isolates and the cellular events in the
transition of the mould phase to the yeast phase (Chan and Chow, 1990; Garrison
and Boyd, 1973; Pracharktam et al., 1992).
2. Differential gene expression
Discovering the regulation of Penicillium marneffei phase-specific genes might elucidate the control of its morphogenesis. Several genes that are expressed predominantly
in the yeast form of Penicillium marneffei have been identified. Malate synthaseencoding gene was found to up-regulate in the yeast phase of Penicillium marneffei
as an alternative energy producing pathway (Cooper Jr and Haycocks, 2000). Pongpom et al. (2005) isolated and characterized a catalase–peroxidase protein-encoding
gene (cpeA), which was expressed at a high level during yeast phase formation. Other
genes possibly involved in regulating the dimorphic transition were also studied, e.g.
abaA (Borneman et al., 2000), cflA (Boyce et al., 2001) and TupA (Todd et al.,
2003): the former appears to control the coupling of nuclear and cellular division in
prearthroconidial cells, while the latter two control the correct morphogenesis of yeast
cells. However, none of these have a detectable effect on dimorphic transition (Liu
Special Issue: Biotech Conference
39
et al., 2007).
Investigation of differential gene expression during transition from the mycelial
form to the yeast form of P. marneffei may lead to the discovery of candidate genes
for pathogenicity. By application of suppression subtractive hybridization combined
with real-time semi-quantitative RT-PCR, Liu et al. (2007) identified 43 differentially
expressed genes. These genes were sorted into broad functional categories including cell wall synthesis, signal transduction, cell cycle, substance transport, general
metabolism and stress response, etc. This suggests that a very complex series of
molecular mechanisms is involved in the switching process of P. marneffei (Liu et al.,
2007).
3. Actin expression
Actins are conserved eukaryotic proteins present mainly in the cell cytoplasm. They
are essential cytoskeletal componentsof all fungi, involved in several cellularprocesses.
Actin-encoding genes from a number of pathogenic fungi have been isolated and characterized.
In the study by Kummasook et al. (2011), the structure of the actin-encodinggene
of P. marneffei was identified at both the nucleotideand amino acid levels. The number of nucleotides and amino acids for an open reading frame of actA in P. marneffei
is similar to that of other fungal actins. Moreover, actA contains all the recognized
conserved sequence motifs of actin protein sequences. The expression of actin genes
has been studied in several pathogenic, dimorphic fungi. In Sporothrix schenckii
andHistoplasma capsulatum, studies have demonstrated that the levels of actin gene
transcripts vary during phase transition (el Rady and Shearer Jr, 1997; Han-Yaku,
1996). Similarly, in Paracoccidioides brasiliensis actin transcripts were found to be
prevalent in the yeast form, but decreased during the transition from yeast to mold
(Niño Vega et al., 2007).
The results of the study by Kummasook et al. (2011) suggest that the actinencoding gene in P. marneffei – actA, increases in expression during the initial stages
of morphogenesis and phase transition, but subsequently is transcribed at a steady
state during normal mycelial and yeast growth. This relatively stable level of expression appears to be independent of the stress response in P. marneffei. Hence,
actA transcription appears to be a suitable standard to serve as an internal control
during quantitative analysis of gene expression in P. marneffei for a short period of
incubation (30 min) under stress conditions and for longer incubation periods (2–4 h)
during macrophage infection with conidia (Kummasook et al., 2011).
4. Protein profiling
As mentioned before, no specific genes have been identified in P. marneffei that
strictly induce mould-to-yeast phase conversion. Chandler et al. generated protein
profiles from the P. marneffei yeast and mould phases to help identify potential gene
products associated with morphogenesis. Whole cell proteins from the early stages
of P. marneffei mould and yeast development were resolved by two-dimensional gel
electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid
chromatography-nanospray tandem mass spectrometry. Putative identifications were
40
PhD Interdisciplinary Journal
derived by searching available databases for homologous fungal sequences. Proteins
found common to both mould and yeast phases included the signal transduction
proteins cyclophilin and a RACK1-like ortholog, as well as those related to general
metabolism, energy production, and protection from oxygen radicals. Many of the
mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase
comprised those involved in heat-shock responses, general metabolism, and cell-wall
biosynthesis, as well as a small GTPase that regulates nuclear membrane transport
and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA
protein sequence, which contained the signature motif of Ran-GTPases, exhibited
90% homology to homologous Aspergillus proteins (Chandler et al., 2008).
The study by Chandler et al. (2008) demonstrated the utility of proteomic approaches to studying dimorphism in P. marneffei. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the
molecular mechanisms of phase transition. Finally, the documented increased levels of
RanA expression suggest that cellular development in P. marneffei involves additional
signaling mechanisms than have been previously described (Chandler et al., 2008).
References
Borneman, A. R., M. J. Hynes and A. Andrianopoulos (2000), ‘The abaa homologue
of penicillium marneffei participates in two developmental programmes: conidiation
and dimorphic growth’, Mol Microbiol 38(5), 1034–1047.
Boyce, K. J., M. J. Hynes and A. Andrianopoulos (2001), ‘The cdc42 homolog of
the dimorphic fungus penicillium marneffei is required for correct cell polarization
during growth but not development.’, J Bacteriol 183(11), 3447–3457.
Chan, Y. F. and T. C. Chow (1990), ‘Ultrastructural observations on penicillium
marneffei in natural human infection’, Ultrastruc Pathol 14, 439–452.
Chandler, J. M., E. R. Treece, H. R. Trenary, J. L. Brenneman, T. J. Flickner, J. L.
Frommelt, Z. M. Oo, M. M. Patterson, W. T. Rundle, O. V. Valle, T. D. Kim,
G. R. Walker and C. R. Cooper Jr. (2008), ‘Protein profiling of the dimorphic,
pathogenic fungus, penicillium marneffei’, Proteome Sci 6.
Cheng, N. C., W. W. Wong, C. P. Fung and C. Y. Li (1998), ‘Unusual pulmonary manifestations of disseminated penicillium marneffei infection in three aids patients’,
Med Mycol 36, 429–432.
Cooper Jr, C. R. and N. G. Haycocks (2000), ‘Penicillium marneffei: an insurgent
species among the penicillia’, J Eukaryot Microbiol 47(1), 24–28.
el Rady, J. and G. Shearer Jr (1997), ‘Cloning and analysis of an actin-encoding cdna
from the dimorphic pathogenic fungus histoplasma capsulatum’, J Med Vet Mycol
35(3), 159–166.
Garbino, J., L. Kolarova, D. Lew, B. Hirschel and P. Rohner (2001), ‘Fungemia in
hiv-infected patients: A 12-year study in a tertiary care hospital’, Aids Patient
Care Stds 15, 407–410.
Garrison, R. G. and K. S. Boyd (1973), ‘Dimorphism of penicillium marneffei as
observed by electron microscopy’, Can J Microbiol 19, 1305–1309.
Han-Yaku, H. et al (1996), ‘Differential expression of the 45 kda protein (actin) during
the dimorphic transition of sporothrix schenckii’, J Med Vet Mycol 34(3), 175–180.
Kummasook, A., A. Tzarphmaag, S. Thirach, M. Pongpom, C. R. Cooper Jr and N.
Vanittanakom (2011), ‘Penicillium marneffei actin expression during phase transition, oxidative stress, and macrophage infection’, Mol Biol Rep 38, 2813–2819.
Liu, H., L. Xi, J. Zhang, X. Li, X. Liu, C. Lu and J. Sun (2007), ‘Identifying differen-
Special Issue: Biotech Conference
41
tially expressed genes in the dimorphic fungus penicillium marneffei by suppression
subtractive hybridization’, FEMS Microbiol Lett 270, 97–103.
Niño Vega, G., C. Pérez-Silva and G. San-Blas (2007), ‘The actin gene in paracoccidioides brasiliensis: organization, expression and phylogenetic analyses’, Mycol Res
111(3), 363–369.
Pongpom, P., Jr C. R. Cooper and N. Vanittanakom (2005), ‘Isolation and characterization of a catalase-peroxidase gene from the pathogenic fungus, penicillium
marneffei’, Med Mycol 43(5), 403–11.
Pracharktam, R., S. Sriurairatna and P. Jayanetra (1992), ‘Morphological variation
in pathogenic strains of penicillium marneffei’, J Med AssocThai 75, 172–179.
Rimek, D. et al. (1999), ‘Disseminated penicillium marneffei infection in an hivpositive female from thailand in germany’, Mycoses 42(Suppl 2), 25–28.
Todd, R. B., J. R. Greenhalgh, M. J. Hynes and A. Andrianopoulos (2003), ‘Tupa,
the penicillium marneffei tup1p homologue, represses both yeast and spore development’, Mol Microbiol 48(1), 85–94.
WHO (2007), Who case definitions of hiv for surveillance and revised clinical staging and immunological classification of hiv-related disease in adults and children,
Technical report.
Wong, S. S. Y., K. H. Wong, W. T. Hui and S. S. Lee (2001), ‘Differences in clinical and
laboratory diagnostic characteristics of penicilliosis marneffei in human immunodeficiency virus (hiv)- and non-hiv-infected patients’, J Clin Microbiol 39, 4535–4540.