fungal genetics and biologypeople.ysu.edu/~crcooper01/spermidine proof.pdf22 conidiogenesis 23...

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Fungal Genetics and Biology xxx (2013) xxx–xxx

YFGBI 2583 No. of Pages 8, Model 5G

16 August 2013

Contents lists available at ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier .com/locate /yfgbi

Spermidine is required for morphogenesis in the human pathogenicfungus, Penicillium marneffei

1087-1845/$ - see front matter � 2013 Published by Elsevier Inc.http://dx.doi.org/10.1016/j.fgb.2013.08.001

Abbreviations: BHA, brain heart infusion agar; MM, minimal medium; PDA,potato dextrose agar.⇑ Corresponding author. Fax: +66 53 217144.

E-mail address: [email protected] (N. Vanittanakom).1 Present address: Faculty of Medical Technology, Western University, Kanchana-

buri 71170, Thailand.

Please cite this article in press as: Kummasook, A., et al. Spermidine is required for morphogenesis in the human pathogenic fungus, Penicillium maFungal Genet. Biol. (2013), http://dx.doi.org/10.1016/j.fgb.2013.08.001

Aksarakorn Kummasook a,b,1, Chester R. Cooper Jr. b, Akihiko Sakamoto c, Yusuke Terui c, Keiko Kashiwagi c,Nongnuch Vanittanakom a,⇑a Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailandb Center for Applied Chemical Biology and Department of Biological Sciences, Youngstown State University, One University Plaza, Youngstown, OH 44555, United Statesc Faculty of Pharmacy, Chiba Institute of Science, Choshi, Chiba 288-0025, Japan

a r t i c l e i n f o

293031323334353637383940

Article history:Received 9 March 2013Accepted 1 August 2013Available online xxxx

Keywords:Penicillium marneffeiConidiogenesisDimorphismS-adenosylmethionine decarboxylaseSpermidinePolyamines

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a b s t r a c t

Penicillium marneffei is a thermally dimorphic fungus that is a highly significant pathogen of immunecompromised persons living or having traveled in Southeast Asia. When cultured at 25 �C, the wild-typestrain of P. marneffei exhibits a mycelial morphology that is marked by the development of specializedstructures bearing conidia. Incubation of the wild type at 37 �C, however, promotes the developmentof a yeast form that divides by fission. Development of the yeast morphology in vivo appears to be req-uisite for pathogenesis. In a prior study using Agrobacterium-mediated transformation for random muta-genesis via T-DNA integration, we generated a morphological mutant (strain I6) defective in conidiation.The T-DNA insertion site in strain I6 was determined to be within the gene encoding S-adenosylmethi-onine decarboxylase (sadA), an enzyme critical to spermidine biosynthesis. In the present study, we dem-onstrated that strain I6 was able to grow on rich media in either the mold or yeast forms at 25 �C and37 �C, respectively. However, reduced growth of strain I6 was observed on minimal medium at eithertemperature. In addition, strain I6 produced mycelia with impaired conidiation on minimal medium at25 �C. Supplementation of minimal medium with spermidine restored the ability of strain I6 to produceconidia at 25 �C and promoted yeast development at 37 �C. Moreover, conidia of strain I6 exhibited poorgermination frequencies in the absence of this polyamine. All three of these processes (conidiogenesis,germination, and growth) were reinstated in strain I6 by complementation of the partially deleted of sadAgene by ectopic insertion of an intact wild-type copy. These results augment prior observations that sper-midine biosynthesis is essential to normal growth, conidiogenesis, spore germination, and dimorphism ina variety of fungi. Given the presumption that P. marneffei infections are initiated following inhalation ofconidia, and that pathogenesis is dependent upon yeast development, this study further suggests that thespermidine biosynthetic pathway may serve as a potential target for combating infections by this med-ically important fungus.

� 2013 Published by Elsevier Inc.

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1. Introduction

Penicillium marneffei is a thermally dimorphic fungus and ahighly significant pathogen of immune compromised persons liv-ing or having traveled in Southeast Asia (Boyce and Andrianopou-los, 2013; Vanittanakom et al., 2006). When cultured at 25 �C, P.marneffei exhibits a mycelial morphology that is marked by thedevelopment of specialized structures bearing conidia. Incubation

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at 37 �C, however, promotes the development of a yeast form thatdivides by fission. The latter morphology is characteristic of the tis-sue phase of P. marneffei. Previously, using an Agrobacterium-med-iated transformation system, we generated a mutant (strain I6) ofP. marneffei that grew as a yeast at 37 �C, but exhibited non-spor-ulating mycelia at 25 �C (Kummasook et al., 2010). Subsequentanalysis suggested that strain I6 possesses a disruption mutationin the gene, designated sadA, encoding S-adenosylmethioninedecarboxylase. This enzyme plays a key role in polyamine biosyn-thesis (Ruiz-Herrera, 1994; Tabor and Tabor, 1985).

Polyamines are polycationic molecules that have been impli-cated in a wide variety of biological reactions (Igarashi and Kash-iwagi, 2010; Ruiz-Herrera, 1994; Tabor and Tabor, 1985). Thethree most common polyamines found in microorganisms are

rneffei.

http://dx.doi.org/10.1016/j.fgb.2013.08.001

mailto:[email protected]


http://www.sciencedirect.com/science/journal/10871845

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putrescine, spermidine, and spermine. These polyamines are syn-thesized in a well characterized pathway in which putrescine isformed from orinithine (Fig. 1A). Subsequently, aminopropylgroups from decarboxylated S-adenosylmethione are added toform spermidine and spermine. The latter group transfer reactionsare critical steps in polyamine biosynthesis that would not be pos-sible without the action of the enzyme S-adenosylmethioninedecarboxylase, a product of the sadA gene.

Numerous studies have documented that polyamines areimportant in fungal cell differentiation processes including sporu-lation, spore germination, colonization, and dimorphism (Chenget al., 2012; Guevara-Olvera et al., 1993, 1997, 2000; Herreroet al., 1999; Jimenez-Bremont et al., 2001; Khurana et al., 1996;Lopez et al., 1997; Reyna-Lopez and Ruiz-Herrera, 2004; San-Blaset al., 1996; Valdés-Santiago et al., 2009, 2012a,b). For example,in a S-adenosylmethionine decarboxylase mutant of the dimorphicfungus Ustilago maydis, recent investigations demonstrated thatlow spermidine concentrations sustained mycelial growth, butyeast development required higher concentrations of this poly-amine (Valdés-Santiago et al., 2012b). Moreover, this mutant wasshown to be avirulent in a plant infection model. By comparison,in the arbuscular mycorrhizal fungus Glomus etunicatum, poly-amines not only stimulated hyphal branching and spore germina-tion, but also appeared to play a critical role in establishingsymbiosis with plant roots (Cheng et al., 2012). As a third example,deletion of the spermidine synthase (spdA) gene in Aspergillus nidu-lans inhibited conidiogenesis (Jin et al., 2002). Supplementation ofthe DspdA strain with appropriate levels of spermidine restoredconidia formation.

In this report, we employ both biochemical and reverse genetictechniques to document the critical role of the P. marneffei sadAgene in vegetative growth, conidiogenesis, conidial germination,and dimorphism. These observations have pertinent implicationsin understanding virulence given the presumption that P. marneffeiinfections are initiated following inhalation of conidia and that thedevelopment of the yeast phase is requisite for the developmentand persistence of infections by this fungus (Vanittanakom et al.,2006). Our collective observations further suggest that the poly-amine biosynthetic pathway may serve as a potential target forcombating pathogenesis of P. marneffei.

2. Materials and methods

2.1. Strains and maintenance

The wild-type strain of P. marneffei (strain F4 [sadA+]; CBS119456) (Pongpom et al., 2005), mutant strain I6 (DsadA) (Kumma-sook et al., 2010), and a genetically complemented transformant

Fig. 1. (A) Polyamine biosynthetic pathway indicating the particular reaction controlledwild-type strain (F4) grown at 25 �C on PDA in the presence or absence of spermidine.

Please cite this article in press as: Kummasook, A., et al. Spermidine is requiredFungal Genet. Biol. (2013), http://dx.doi.org/10.1016/j.fgb.2013.08.001

(strain C3; sadA+/DsadA) were routinely maintained on malt ex-tract agar (Oxoid, Hampshire, United Kingdom) incubated at25 �C. For long-term maintenance, strains were stored as previ-ously described (Chandler et al., 2008).

2.2. Reagents and media

Unless otherwise noted, all chemical reagents and antibioticswere purchased from Amresco (Solon, OH). Oligonucleotide prim-ers were obtained from Integrated DNA Technologies (Coralville,IA). The polyamines putrescine, spermidine, and spermine werepurchased from MP Biomedicals (Solon, OH).

Nutrient-limited media employed in these investigations in-cluded potato dextrose agar (PDA [Difco brand] Becton Dickinson,Franklin Lakes, NJ) and minimal medium agar ([MM] 0.2% NH4Cl;0.1% (NH4)2SO4; 0.05% KCl; 0.05% NaCl; 0.1% KH2PO4; 0.05% MgSO4-

�7H2O; 0.002% FeSO4�7H2O; 1% glucose; 1.5% agar). Brain–heartinfusion agar (BHA [Difco brand]; Becton Dickinson) served asthe nutrient-rich medium in this study.

2.3. Molecular techniques and analyses

DNA extraction procedures, Southern blot analyses, and inversepolymerase chain reaction (PCR) protocols have been describedpreviously (Kummasook et al., 2010). Oligonucleotide primerswere designed using Primer3 [(Rozen and Skaletsky, 2000);http://frodo.wi.mit.edu/primer3/input.htm]. Analyses of DNA se-quences were performed using the BLAST [(Altschul et al., 1990,1997); http://blast.ncbi.nlm.nih.gov/Blast.cgi] and Geneious (ver-sion 6.06, BioMatters, Ltd., Aukland, New Zealand; http://www.geneious.com) programs in conjunction with the P. marneffeigenome database (GenBank Accession Number ABAR00000000).

2.4. Complementation of strain I6

A 3756-bp fragment covering the sadA open reading frame,including 1672-bp fragment of the 50 flanking sequence and 573-bp of the 30 flanking sequence, was amplified from the wild typeof P. marneffei (strain F4) in a PCR using primers sadF-NgoMIV(50-CAGCAGCCGGCACATTGAATATTAAGACGA-30; underlined se-quence is the NgoMIV restriction site) and sadR-SbfI (50-CAGATCCTGCAGGTGCTTGGCTTGATTGATTA-30; underlined se-quence is the SbfI restriction site). Amplification reactions (20 lltotal volume) were performed in an MJ Mini Personal Thermal Cy-cler (Bio-Rad, Hercules, CA) using Phusion� Hot Start High-FidelityDNA Polymerase (Finnzymes, Espoo, Finland) in accord with thevendor’s instructions. The parameters of the PCR were as follows:98 �C for 30 s; 35 cycles of 98 �C for 10 s, 60 �C for 30 s, and 72 �C

by the sadA gene product. (B) Colony morphologies of the sadA mutant (I6) and the

for morphogenesis in the human pathogenic fungus, Penicillium marneffei.

http://frodo.wi.mit.edu/primer3/input.htm

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://www.geneious.com

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for 3 min; 72 �C for 10 min; and hold at 4 �C. The PCR productswere ethanol-precipitated (Gallagher and Wiley, 2008) prior todigestion with restriction enzymes NgoMIV and SbfI (New EnglandBiolabs, Ipswich, Massachusetts). The digested PCR products werepurified by agarose gel extraction using the QIAquick Gel Extrac-tion Kit (Qiagen, Valencia, CA), then ligated into NgoMIV-/SbfI-trea-ted pAN7-1 plasmid (Punt et al., 1987) using T4 DNA ligase (NewEngland Biolabs, Ipswich, MA). The resulting plasmid, designatedpAN7.1-sadA, also contains the gene, hph, which confers resistanceto the antimicrobial agent, hygromycin B. This hybrid vector wasmaintained in Escherichia coli DH5a following transformation intocompetent cells of this bacterial strain (Gallagher and Wiley, 2008).For genetic complementation of the P. marneffei DsadA mutant, theplasmid was isolated from this bacterium using the method ofSambrook and Russell (2001).

For complementation studies, protoplasts of P. marneffei strainI6 (DsadA) were transformed with plasmid pAN7.1-sadA followinga previously described protocol (Borneman et al., 2001). The trans-formed protoplasts were plated onto BHA containing hygromycin B(200 lg/ml) and 1.2 M sorbitol. The plates were incubated at 25 �Cfor 5 days before being transferred to 37 �C for an additional5 days. Putative transformants arose as yeast-like colonies. Drug-resistant clones of these transformants were isolated by streakingportions of separate colonies onto BHA/hygromycin media(without sorbitol) followed by incubation at 37 �C. Subsequently,positive transformants were identified from selected hygromy-cin-resistant isolates through (i) PCR amplification of the intactsadA gene using primers sadF-NgoMIV and sadR-SbfI, and (ii)Southern blot analysis using pAN7-1 as a probe (data not shown).PCR positive clones possessing a single integrated copy of pAN7.1-sadA were subjected to further analysis. Complementation of thesadA� phenotype in these clones was confirmed by growth of con-firmed transformants on nutrient-limited media (PDA or MM) inthe absence of exogenous spermidine.

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2.5. Phenotypic screening of P. marneffei strains

2.5.1. Conidial inoculum preparationStrains F4 (sadA+), I6 (DsadA), and C3 (sadA+/DsadA) were cul-

tured on PDA containing 3 mM of spermidine for 7 days at 27 �C.Following the scraping of surface growth and the suspension ofmycelia in sterile saline, conidia were isolated by filtration throughsterile glass wool (Gifford and Cooper, 2009). Isolated conidia werecounted using a hemocytometer chamber.

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2.5.2. Drop dilution assaysFor drop dilution assays, a series of ten-fold dilutions were pre-

pared from each strain at a starting solution of 1 � 108 conidia/ml.From these suspensions, 5 ll aliquots were spotted onto MM agarplates supplemented with or without 3 mM spermidine. Theseplates were subsequently incubated at 27 �C, 37 �C, or 39 �C for4 days.

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2.5.3. Measurement of colonial growthTo measure the radial growth of colonies from each strain, sus-

pensions of each strain were prepared containing 1 � 106 conidia/ml. For a given strain, 3 ll of this suspension was spotted at threedifferent, equal distance locations on a 90 mm Petri dish contain-ing MM agar with or without the following concentrations of sper-midine: 0.00, 0.05, 0.10, 0.50, 1.00, 2.00, and 3.00 mM. The plateswere then incubated at 27 �C and the diameters (in mm) of eachresulting colony were measured at 24 h intervals over a 2–7 dayperiod.


2.5.4. ConidiogenesisTo assess the degree of conidiogenesis among the various

strains, 3 ll of a 106 conidia/ml suspension was spotted in the cen-ter of MM agar plates supplemented with one of the following con-centrations of spermidine: 0.00, 0.05, 0.10, 0.50, 1.00, 2.00, and3.00 mM. At every concentration, six plates were inoculated forall three strains studied. The plates were then incubated at 27 �C.After 48 h of incubation, and every 24 h interval thereafter, oneplate of each strain cultured on the different spermidine supple-mented media was chosen from which to collect conidia. The con-idia from the resulting colonies were harvested by scraping thecolony surface using a cotton swab. The conidia collected in thismanner were suspended in known volume of normal saline (0.9%NaCl) containing 0.05% (v/v) Tween 20, then counted using ahemocytometer. The total number of conidia collected from eachcolony was calculated. These assays were performed in triplicateand conidial counts were analyzed using standard t-tests (Graph-Pad Prism, version 5; GraphPad Software, La Jolla, CA).

2.5.5. Conidial germinationApproximately 1 � 106 conidia, isolated from each strain as de-

scribed above, were used to inoculate 24-well plates with eachwell containing 300 ll of culture broth (MM without agar) supple-mented with one of the following concentrations of spermidine:0.00, 0.05, 0.10, 0.50, 1.00, 2.00, and 3.00 mM. The plates wereincubated at 27 �C or 37 �C prior to determination of germinationfrequency. The germination rate was measured microscopicallyby counting the number of germinating conidia (conidia with a vis-ible germ tube) in populations of at least 100. Three independentexperiments were performed. The means and standard errors ofthe mean values were calculated using GraphPad Prism.

2.6. Measurement of polyamines

Polyamine concentrations in the wild-type (sadA+) and mutantI6 (DsadA) strains of P. marneffei were determined from MM brothcultures inoculated with 106 conidia/ml, then incubated at either25 �C or 37 �C for 5 days. The strain I6 cultures were supplementedwith 0.1 mM spermidine. The fungal cells from these cultures wereharvested by centrifugation, washed twice with saline, then sus-pended in 10% trichloroacetic acid. Following mechanical shakingfor 2 min, the suspensions were incubated at 70 �C for 30 min. Sub-sequently, the suspensions were subjected to centrifugation. Theresulting supernatant was removed and the polyamine contentdetermined by high-pressure liquid chromatography as describedpreviously (Igarashi et al., 1986). In addition, the protein contentof the precipitate was quantified by the method of Bradford (1976).

3. Results

3.1. Isolation and genetic characterization of a sadA mutant

Strain I6 was originally isolated as a bleomycin-resistant, yeast-like colony growing at 37 �C on bleomycin-supplemented BHA fol-lowing random insertional mutagenesis by Agrobacterium-medi-ated transformation (Kummasook et al., 2010). When cultured at25 �C on PDA, the mutant grew slowly and appeared defective inconidiation (Fig. 1B). In contrast, the wild type (strain F4), pro-duced rapidly growing, heavily conidiated colonies on PDA at25 �C (Fig. 1B).

Inverse PCR of XhoI-digested DNA from strain I6 generated a1.5 kbp product, but no amplification product was derived fromthe wild type (Fig. 2A), thereby indicating successful T-DNA inte-gration. Southern blot analysis of strain I6 was performed using afragment of the bleomycin-resistance gene (ble) as a probe. The re-



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sults confirmed that the mutation in strain I6 resulted from a sin-gle T-DNA integration event (Fig. 2B). Subsequent sequencing andanalysis of the nucleotides flanking the integration site revealedthat the gene encoding S-adenosylmethionine decarboxylase(sadA; GenBank Accession No. XM_002147750) was disruptedand a small portion deleted (Fig. 2C). Specifically, 806 bp of the 50

end of sadA and 1169 bp of non-coding region locating upstreamof the gene were removed (data not shown). Hence, strain I6 wasgiven the genetic designation of DsadA.

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3.2. Polyamine requirement of strain I6 (DsadA)

Because the sadA product has been shown in other fungi to havea crucial role in polyamine biosynthesis (Fig. 1), the phenotype ofstrain I6 grown in the presence of selected pathway intermediateswas evaluated. On PDA or MM agar, growth of strain I6 was limitedand virtually no conidia were produced. Similarly, supplementa-tion of the medium with putrescine did not restore growth or con-idiogenesis (data not shown). However, the addition of 3 mMspermidine restored growth and conidiogenesis to a significant de-gree (Fig. 1B). Curiously, spermine supplementation increasedgrowth and conidiation, but not nearly to the degree of that pro-duced by the addition of spermidine to the medium (data notshown). In contrast, supplementation of media with putrescine,spermidine, or spermine had no observable affect upon the wild-type strain of P. marneffei (Fig. 1B; data not shown).

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Fig. 2. Inverse PCR, Southern blot, and sequence analysis of the sadA mutant. (A) Anapproximately 1.5 kilobase (kb) PCR product fragment was derived from an inversePCR when using XhoI-digested genomic DNA from I6 mutant, whereas XhoI-digested genomic DNA from wild type (F4) was used as a negative control. Lane Mcontains a 1 kb ladder as size standard with specific bands noted on the left. (B)Southern blot analysis was performed using genomic DNA from sadA mutant, whichwas digested with NdeI (Lane1) or KpnI (Lane 2), and able gene fragment as a probe(Kummasook et al., 2010). The hybridization results show single bands in each laneindicating the lone integration of T-DNA in the I6 mutant. The absence of anyhybridization signal in NdeI-digested DNA from the wild type is shown in Lane 3.Lane 4 depicts the positive control, which consists of a 752 bp ble gene fragment.The position of the molecular size markers, in base pairs (bp) is shown on the left.(C) Sequence analysis revealed that T-DNA integration of I6 mutant leads to partialdeletion of the S-adenosylmethionine decarboxylase-encoding gene. This is dia-grammatically shown here in which the components include a triangle depictingthe T-DNA integration site and an arrow indicating the 50-to-30 direction of theremaining portion of the sadA gene. The thick line represents flanking genomicDNA, whereas the thin line signifies deleted DNA sequences. This figure is notwholly to scale.


3.3. Genetic complementation of strain I6 (DsadA)

A full-length open reading frame of the P. marneffei sadA genewas cloned into plasmid pAN7.1. This construct was used to trans-form protoplasts of strain I6 via ectopic integration of the hybridvector. Three transformants were selected independently as hygro-mycin-resistant clones. All showed growth, the ability to produceabundant numbers of conidia when cultured on MM or PDA notcontaining spermidine (data not shown). One of these transfor-mants, C3 (sadA+/DsadA), was selected for comparative studiesagainst the wild type (strain F4; sadA+) and strain I6 (DsadA).

3.4. Spermidine is critical to growth and conidiogenesis

The effect of spermidine on growth at 27 �C, 37 �C, and 39 �Cwas assessed using a spotting assay (Fig. 3). After 7 days on MMalone, strain I6 exhibited very little growth to no growth at allinoculum concentrations and incubation temperatures. Since theremaining spermidine added to the pre-culture medium in theconidia, the mutant showed the slight growth in some concentra-tions of inocula. In fact, virtually no growth was detected on platesincubated at 39 �C. By comparison, the wild type and comple-mented strain C3 grew quite well at all inoculum concentrationsat 27 �C and 37 �C, but displayed reduced growth at 39 �C. Similarexperiments were conducted on MM agar supplemented with3 mM spermidine. The growth of strain I6 appeared similar to thatof strains F4 and C3 at all inoculum concentrations and incubationtemperatures.

To assess the importance of spermidine to conidial production,strains F4, I6, and C3 were cultured for 7 days at 27 �C on MM agarsupplemented with varying concentrations of this polyamine. Thecolony morphologies of strains F4 and C3 appeared similar regard-less of the spermidine concentration, whereas strain I6 required3 mM spermidine to acquire a gross wild type-like colony pheno-type (Fig. 4). Moreover, while the diameters of the I6 colonies gen-erally paralleled the increase in spermidine concentration, fewerconidia were produced compared to those generated by the wild-type and complemented strains (Table 1). The difference in conid-ial production was significantly different between the wild typeand DsadA mutant at all levels of spermidine supplementation(p = 0.05). Curiously, increased spermidine concentrations tendedto decrease conidial production by strains F4 and C3 up to a thresh-old level of 3 mM, whereupon the number of conidia produced in-creased significantly.

3.5. Spermidine is crucial to conidial germination

The germination frequencies of conidia from strains F4, C3, andI6 were determined. In MM liquid medium, the percentage of ger-minated conidia from strains F4 and C3 were essentially equal ateach time point when incubated at 27 �C or 37 �C (Fig. 5). After24 h of incubation, however, conidia from these strains incubatedat 37 �C germinated at less than half the rate of those incubatedat 27 �C. Also, germination of strain F4 and C3 conidia first occurredbetween 8–12 h of incubation at 27 �C and between 12–16 h incu-bation at 37 �C. In contrast, I6 conidia germinated approximately20 h after incubation at 27 �C. Moreover, the percentage of germi-nated conidia after 24 h was about four times less than those ofstrains F4 and C3. At 37 �C, conidia of strain I6 exhibited little, ifany germination after 24 h of incubation.

The effect of spermidine supplementation on conidial germina-tion was examined as well. Exogenous spermidine was added tothe liquid MM at different concentrations and the germination fre-quency of conidia from strains F4, C3, and I6 was measured after20 h of incubation at 27 �C and after 24 h of incubation at 37 �C(Fig. 6). Under both conditions, the germination frequencies of



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Fig. 3. Drop dilution assay. Series of ten-fold dilutions of the wild type (F4; sadA+), mutant (I6; DsadA), and complemented (C3; DsadA/sadA+) strains, derived from a startingsolution of 1 � 108 conidia/ml, were spotted in aliquots of 5 ll onto MM plates with or without 3 mM spermidine. The plates were subsequently incubated for 4 days at 27 �C(A and D), 37 �C (B and E), or 39 �C (C and F). Growth in the absence of spermidine is shown in A–C, whereas growth in the presence of spermidine is depicted in D–F.

Fig. 4. Affect of spermidine on colony size in Penicillium marneffei. 3 � 103 conidia of the wild type (F4; sadA+), mutant (I6; DsadA), and complemented (C3; DsadA/sadA+)strains were inoculated onto MM plates supplemented with spermidine at various concentrations: 0.00, 0.05, 0.10, 0.50, 1.00, 2.00, and 3.00 mM. The plates were incubated at27 �C for 7 days.

Table 1Effect of different spermidine concentrations on the growth and conidiation of the Penicillium marneffei wild type (F4), the DsadA mutant (I6), and the complemented strain (C3)a.

Spermidine (mM) Diameters (cm)a Spores (106/cm2)b

F4 I6 C3 F4 I6 C3(sadA+) (DsadA) (DsadA/sadA+) (sadA+) (DsadA) (DsadA/sadA+)

0.00 2.20 0.50 2.20 134.0 0.00 121.30.05 2.30 1.80 2.30 118.6 1.32 103.60.10 2.20 1.80 2.20 88.5 1.49 87.10.50 2.20 1.90 2.20 96.2 2.69 80.41.00 2.20 2.00 2.20 80.8 4.32 89.22.00 2.20 2.00 2.20 57.7 10.12 63.73.00 2.15 1.90 2.15 101.0 19.50 95.3

a Data was collected after 7 days of incubation at 27 �C on minimal medium with and without various concentrations of spermidine.b Values are means of three replicates. Means were analyzed by t-test for each concentration of spermidine. The spore counts of strain I6 were significantly different from

the wild type (F4) and the complemented strain (C3) at every concentration of spermidine (P = 0.05).



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strains F4 and C3 were essentially identical not only to each other,but also remained nearly the same at each concentration of sper-midine. By comparison, strain I6 exhibited an increase in germi-nated conidia at both temperatures that paralleled the increasingspermidine concentration. At 3 mM spermidine, the percentageof germinated conidia from strain I6 incubated at 27 �C or 37 �Cwas nearly the same as that exhibited by strains F4 and C3.


3.6. Polyamine levels of mycelium and yeast phase

The polyamine content of the wild type cultured in MM for5 days at 25 �C was higher than parallel cultures incubated at37 �C (Fig. 7). Spermidine was the most abundant free polyaminein both mycelial and yeast cells, although both putrescine andspermine were detected at low levels. In contrast, however, putres-



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Fig. 5. sadA affects conidial germination at 27 �C and 37 �C. Conidia (1 � 106) from the wild-type (F4; sadA+), mutant (I6; DsadA), and complemented (C3; DsadA/sadA+)strains were grown in MM broth at 27 �C or 37 �C. The percentage of germinated conidia was measured after 4, 8, 12, 16, 20 and 24 h of incubation by counting at least 100conidia per treatment. Three independent experiments were performed and standard error bar of the mean is shown for each time point.

Fig. 6. Spermidine is required for conidial germination at both 27 �C and 37 �C. Conidia (1 � 106) from the wild type (F4; sadA+), mutant (I6; DsadA), and complemented (C3;DsadA/sadA+) strains were grown in MM broth with or without spermidine at different concentrations. The percentage of germinated conidia was measured after 20 h ofincubation at 27 �C (A) and after 24 h of incubation at 37 �C (B). Three independent experiments were performed and a standard error bar of the mean is shown for eachexperimental condition.



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cine was the most abundant polyamine extracted from 25 �C cul-tures of the DsadA mutant (strain I6), with spermidine supplemen-tation. At 37 �C, putrescine levels decreased significantly, butremained higher than the wild type cultured in MM alone at eithertemperature.

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Fig. 7. Polyamine content of P. marneffei cells. Wild-type cultures were incubatedwithout exogenous spermidine for 5 days at 25 �C (Sample No. 1) or 37 �C (SampleNo. 2). The mutant I6 strain was similarly cultured at 25 �C (Sample No. 3) and 37 �C(Sample No. 4) in media containing 0.1 mM spermidine. The polyamine concen-trations are shown as nmol/mg protein. PUT, putrescine; SPD, spermidine; SPM,spermine.

4. Discussion

Among the fungi, spermidine is a critical polyamine havingimportant roles in growth and development. For example, spermi-dine is an essential metabolite for normal growth in Neurosporacrassa (Pitkin and Davis, 1990). Also, spermidine appears essentialfor dimorphism in U. maydis (Valdés-Santiago et al., 2012b). Fur-thermore, Jin et al. (2002) have proposed that several intracellularspermidine threshold levels exist in A. nidulans that regulate differ-ent developmental or physiological processes.

In the present study, we demonstrated that spermidine is re-quired for conidiogenesis in P. marneffei. The disruption mutant,strain I6 (DsadA), exhibited an inability to produce significantnumbers of conidia on both PDA and MM plates, even at a latestage of culture. However, conidiogenesis could be restored whenthe growth medium was supplemented with various concentra-tions of spermidine (Fig. 4 and Table 1). The number of conidia in-creased as the concentration of spermidine increased, suggestingthat this polyamine is required for conidiation in a dose-dependentmanner (Table 1). These observations are further supported by theapparently full restoration of conidiation in strain C3 following ec-topic insertion of the sadA gene in a S-adenosylmethionine decar-boxylase mutant (strain I6). Notably, though, the number ofconidia produced by strain I6 on media supplemented with


3 mM spermidine were still significantly lower than those pro-duced by the wild-type strain (F4) and the DsadA-complementedstrain (C3) on the same medium. The reason that the level of con-idiation in strain I6 remained significantly lower than strains F4and C3 on media supplemented with spermidine is not entirelyclear. Perhaps a greater threshold level of the exogenously suppliedpolyamine is required to achieve full conidial production. Conceiv-ably, the sadA gene is functionally impacting the efficiency of



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conidiogenesis through other metabolic or signaling pathways inaddition to spermidine biosynthesis. Hence, supplying exogenousspermidine only partially compensates for the absence of this genein the DsadA mutant.

We also observed a general decline in the number conidia instrains F4 and C3 with increasing spermidine concentrations, butthe mechanism is unknown (Table 1). Additionally, spermidinehas apparently effects on the wild type as well as complementingdefects in the sadA mutant by suppressing production of red pig-ment associated with the mold form. It has been reported thatthe yellow pigment of Penicillium chrysogenum and other metabo-lites are influenced the spermidine-mediated LaeA regulator (Mar-tín et al., 2012). Because P. marneffei contains an laeA gene(GenBank Accession No. XM_002146873.1), suppression of red pig-ment production may involve a LaeA-mediated mechanism.

Moreover, it is interesting to note that Jin et al. (2002) observedthat overexpression of the gene flbA helped promote conidiogene-sis at lower levels of spermidine. The flb genes are critical to nor-mal sporulation processes in Aspergillus spp. (Ni et al., 2010). Thisobservation supports the investigators’ contention that thresholdlevels of polyamines are required for step-wise progression ofdevelopmental processes. In addition, similar threshold levels werepostulated to promote dimorphism in the plant pathogen, U. may-dis (Guevara-Olvera et al., 1997). It is possible that the same phe-nomenon exists in P. marneffei, a fungus that undergoesconidiogenesis as well as conversion from a mold to a yeast form.Experiments similar to those described in A. nidulans and U. maydismay be possible in that an flbD over expression mutant of P. mar-neffei has been isolated (Kummasook et al., 2010; unpublisheddata). Yeast development of the latter mutant is limited at 37 �C.Instead, over expression of flbD in P. marneffei permits the contin-uation of conidiogenesis under normally non-permissive condi-tions (unpublished observations). This mutant may help toascertain if polyamine threshold levels are essential to conidiogen-esis, conidial germination, and dimorphism.

In addition to conidiogenesis, our investigations demonstratedthat spermidine is essential to conidial germination, growth, anddimorphism in P. marneffei. For conidia of strain I6 incubated inMM broth, germination is restored with exogenously suppliedspermidine. Moreover, strain I6 grows poorly on MM or PDA, butboth mold and yeast growth of this mutant are supported on richor spermidine-supplemented media. By contrast, regardless ofthe medium employed, conidia from strain C3 germinate and growin the absence of this polyamine. Strain C3 will also undergo mold-to-yeast conversion at 37 �C in the absence of spermidine. Theseresults parallel those previously noted in both monomorphicmolds and dimorphic fungi (Guevara-Olvera et al., 1993, 1997,2000; Herrero et al., 1999; Jimenez-Bremont et al., 2001; Jinet al., 2002; Khurana et al., 1996; Lopez et al., 1997; Reyna-Lopezand Ruiz-Herrera, 2004; San-Blas et al., 1996; Valdés-Santiagoet al., 2009).

While our genetic and phenotypic analyses attest to the impor-tance of polyamines in the morphogenesis of P. marneffei, we soughtto support these observations with direct evidence of specific poly-amine levels in the wild-type and mutant strains. In particular, wedemonstrated that wild-type mycelia contain approximately five-fold more spermidine than that of the DsadA mutant (Fig. 7). Con-versely, at 25 �C, the mutant was shown to possess nearlyeighteen-fold higher levels of putrescine than the wild type. At37 �C, although spermidine levels in both the wild type and mutantappeared similar, putrescine was essentially non-detectable in thewild type whereas much higher concentrations were demonstratedin the DsadA mutant. These results are consistent with expectationsof a metabolic block in polyamine biosynthesis due to the absenceor dysfunction of S-adenosylmethionine decarboxylase. By compar-ison, spermine levels in the mutant and wild-type strains were very


low under each of the conditions assayed. Such observations areconsistent with non-detectable amounts of cellular spermine in fil-amentous fungi as well as the dimorphic fungi U. maydis and Para-coccidioides brasiliensis (Nickerson et al., 1977; San-Blas et al.,1996; Valdés-Santiago et al., 2009).

The collective observations of the present study may provideimportant insights into the mechanisms underlying cellular devel-opment in this dimorphic fungus, thus having pertinent implica-tions in the potential prevention and treatment of infectionscaused by P. marneffei. Pathogenesis by P. marneffei is presumablyinitiated via the inhalation of conidia, or even perhaps arthroconi-dia (Vanittanakom et al., 2006). Subsequently, evidence suggeststhat these infectious propagules lodge within the lung where theyare engulfed by phagocytic cells. Once within the harshintracellular environment, conidia that successfully avoid the hostcell’s defense mechanisms undergo phase transition to form thefission-yeast morphology typical of penicilliosis due to P. marneffei.Hence, since spermidine plays a crucial role in cellular growth anddifferentiation in P. marneffei, as do other polyamines in patho-genic, dimorphic fungi (Guevara-Olvera et al., 2000; Herreroet al., 1999; Lopez et al., 1997; San-Blas et al., 1996), then the poly-amine biosynthetic pathway may serve as an attractive target forthe development of antifungal modalities. Moreover, this pathwayemploys several easily studied enzymes that might be exploited inthe development of antifungal strategies. Therefore, based uponthe present and prior studies, further investigations into the roleof polyamines in fungal pathogenesis are warranted.

5. Conclusions

We successfully characterized a conidiation-defective mutant ofP. marneffei generated via Agrobacterium-mediated transformation.This mutant, strain I6, harbors a disruption mutation in the genethat we have designated sadA. This gene encodes S-adenosylmethi-onine decarboxylase and is critical to polyamine biosynthesis, spe-cifically the conversion of putrescine to spermidine. We furtherdemonstrated that spermidine is crucial for growth, conidiogene-sis, conidial germination, and dimorphism in P. marneffei. Ourobservations suggest that the polyamine biosynthetic pathwayrepresents a potential avenue for developing chemotherapeuticinterventions to prevent and treat infections by P. marneffei andpossibly other pathogenic fungi.

Acknowledgments

This work was supported by a Royal Golden Jubilee PhD re-search assistant fellowship to AK from the Thailand Research Fund,the ‘‘National Research University’’ Project of the Thai Ministry ofEducation, and the Faculty of Medicine, Chiang Mai University.The University Research Council and the Office of Student Affairsat Youngstown State University provided additional support. Theauthors also thank Dr. Lorna Gallagher for critically reviewingthe manuscript.

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