Theerapong Krajaejun, Marcel Wüthrich, Gregory M.
Gauthier, et al.
2010. Discordant Influence of Blastomyces
dermatitidis Yeast-Phase-Specific Gene BYS1 on
Morphogenesis and Virulence. Infect. Immun.
78(6):2522-2528.
doi:10.1128/IAI.01328-09.
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Discordant Influence of
Blastomyces dermatitidis
Yeast-Phase-Specific Gene BYS1
on Morphogenesis and Virulence
Vol. 78, No. 6
Discordant Influence of Blastomyces dermatitidis Yeast-Phase-Specific
Gene BYS1 on Morphogenesis and Virulence䌤
Theerapong Krajaejun,1† Marcel Wüthrich,1 Gregory M. Gauthier,2 Thomas F. Warner,3
Thomas D. Sullivan,1* and Bruce S. Klein1,2,3,4
Departments of Pediatrics,1 Medicine,2 Pathology and Laboratory Medicine,3 and Medical Microbiology and Immunology,4
School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706
Received 1 December 2009/Returned for modification 1 January 2010/Accepted 23 March 2010
Blastomyces dermatitidis is a thermally induced dimorphic fungus capable of causing lung and systemic
infections in immunocompetent animal hosts. With the publication of genomic sequences from three different
strains of B. dermatitidis and the development of RNA interference as a gene-silencing tool, it has become
possible to easily ascertain the virulence and morphological effects of knocking down the expression of
candidate genes of interest. BYS1 (Blastomyces yeast-phase-specific 1), first identified by Burg and Smith, is
expressed at high levels in yeast cells and is undetectable in mold. The deduced protein sequence of BYS1 has
a putative signal sequence at its N terminus, opening the possibility that the BYS1-encoded protein is
associated with the yeast cell wall. Herein, strains of B. dermatitidis with silenced expression of BYS1 were
engineered and tested for morphology and virulence. The silenced strains produced rough-surfaced cultures on
agar medium and demonstrated a propensity to form pseudohyphal cells on prolonged culture in vitro and in
vivo, as measured in the mouse lung. Tests using a mouse model of blastomycosis with either yeast or spore
inocula showed that the bys1-silenced strains were as virulent as control strains. Thus, although silencing of
BYS1 alters morphology at 37°C, it does not appear to impair the pathogenicity of B. dermatitidis.
been used as a live attenuated vaccine (26). H. capsulatum
contains two well-studied yeast-phase-specific genes with
pronounced roles in pathogenesis, i.e., that for calcium
binding protein 1 (CBP1) (21) and that for yeast-phasespecific protein 3 (YPS-3) (2, 3). Coccidioides spherule outer
wall glycoprotein (SOWgp) is an immunodominant protein
encoded by a parasitic-phase-specific gene that also has a
role in adhesion and virulence (12). These examples strongly
implicate yeast-phase-specific genes as crucial for virulence
in the endemic dimorphic fungi.
In addition to BAD1 in B. dermatitidis, a second yeast-phasespecific gene, that for Blastomyces yeast-phase-specific protein
1 (BYS1), has been identified by differential cDNA library
screening (4, 9). The 0.9-kb-long BYS1 transcript is highly
expressed in yeast cells but is absent from mold (9). The function of BYS1 remains unknown.
As a means of rapidly analyzing the role of BYS1 in
morphogenesis and virulence, we used a green fluorescent
protein (GFP) sentinel gene-silencing strategy to effectively
suppress BYS1 expression (16). This strategy has been used
to analyze the function of B. dermatitidis genes such as
BAD1 and CDC11, as well as the transgenically expressed
lacZ gene (16). We cloned BYS1 next to GFP in the GFP
sentinel RNA interference (RNAi) vector to create a hairpin to simultaneously silence GFP and BYS1 expression and
allow diminished GFP to report the expression of BYS1.
Strains with decreased expression of BYS1 were thereby
generated and used to study the role of BYS1 in pathogenesis. Although reduced expression of BYS1 promoted the
growth of the organism as pseudohyphae after prolonged
culture in vitro and to a lesser extent in the mouse lung,
bys1-silenced strains showed no alterations in virulence in
vivo in a murine model of infection.
The endemic dimorphic fungi are primary pathogens with
worldwide distribution and include Blastomyces dermatitidis,
Histoplasma capsulatum, Coccidioides immitis, Coccidioides
posadasii, Paracoccidioides brasiliensis, Sporothrix schenckii,
and Penicillium marneffei. Infections caused by these fungi can
cause severe disease in those immunocompromised by AIDS,
malignancy, and pharmacologic immunosuppression (22). Preventive and therapeutic measures to combat these infections
are required, and for this purpose, it is necessary to improve
our understanding of the pathogenesis of these fungi.
The dimorphic fungi can undergo a temperature-dependent
morphological change from conidium or mycelium at environmental temperatures (22°C) to pathogenic yeast at body temperature (37°C) (1, 12, 19–21, 24). B. dermatitidis is endemic to
the Ohio and Mississippi River valleys and causes pneumonia
following the inhalation of conidia from the soil and subsequent conversion to budding yeast cells in animal hosts. Recent
research efforts have focused on identifying genes associated
with this phase transition, based on a hypothesis that such
genes may play important roles in pathogenesis.
Several yeast-phase-specific genes have been identified. In
B. dermatitidis. Blastomyces adhesin 1 (BAD1) (5, 6, 13)
encodes a 120-kDa major surface protein that is expressed
only in the yeast phase and functions as a host cell adhesin
and immunomodulating factor. Deletion of BAD1 attenuates the pathogen’s virulence (7). A bad1-null strain has
* Corresponding author. Mailing address: 1550 Linden Dr., 4301
Microbial Sciences Building, University of Wisconsin, Madison, WI
53706. Phone: (608) 262-7703. Fax: (608) 262-8418. E-mail: tdsulliv
@pediatrics.wisc.edu.
† Present address: Department of Pathology, Faculty of Medicine,
Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
䌤
Published ahead of print on 5 April 2010.
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INFECTION AND IMMUNITY, June 2010, p. 2522–2528
0019-9567/10/$12.00 doi:10.1128/IAI.01328-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
B. DERMATITIDIS BYS1
MATERIALS AND METHODS
Strains and growth conditions. B. dermatitidis strains 26199 (American Type
Culture Collection) and SLH14081 (23) (clinical isolate; referred to here as
14081) were used to generate the GFP reporter strains, 26199-GFP and 14081GFP, used in this study. Both strains are virulent in a murine model of infection.
Strain 55 is a derivative of 26199 in which the virulence factor gene BAD1 was
knocked out by homologous recombination (7). Mycelia of strain 14081, but not
26199, are able to produce conidia. Yeast cells were grown at 37°C on Histoplasma macrophage medium (HMM) (25), Middlebrook 7H10 with oleic acidalbumin-dextrose-catalase enrichment (Becton, Dickinson and Company, Franklin Lakes, NJ), or 3 M medium (25). Mycelial cultures were grown at 22°C on
potato dextrose agar or 3 M medium. Use and maintenance of Agrobacterium
tumefaciens strain LBA1100 were as previously reported (23).
RNA extraction, cDNA synthesis, and bys1 RNAi plasmid constructions. B.
dermatitidis yeast cells were grown in liquid HMM at 37°C for 2 days and
harvested. The yeast cell wall and membrane were disrupted with glass beads
using a Mini-Beadbeater-8 (Biospec Products, Bartlesville, OK). Total RNA was
extracted by using TRI Reagent RT RNA/DNA/Protein reagents according to
the manufacturer’s protocol (Molecular Research Center, Inc., Cincinnati, OH).
RNA was further purified using an RNeasy Mini kit (Qiagen, Valencia, CA); the
concentration was measured spectrophotometrically (Nanodrop Technologies,
Inc., Wilmington, DE), and the integrity was evaluated by agarose gel electrophoresis. To prepare cDNA, 1 ␮g of total RNA was reverse transcribed using
TaqMan reverse transcription reagents and the manufacturer’s protocol (Roche
Molecular Systems, Inc., Branchburg, NJ).
An RNAi vector was constructed to cosilence GFP and BYS1. The BYS1
target sequence was amplified from B. dermatitidis cDNA using gene-specific
primers with the attB1 or attB2 Gateway (Invitrogen, Carlsbad, CA) sequences at the 5⬘ end (primers BdBys1R-AttB1 [5⬘-GGGGACAAGTTTGT
ACAAAAAAGCAGGCTGCCACTGCTTACGCCTGTC-3⬘] and BdBys1FAttB2 [5⬘-GGGGACCACTTTGTACAAGAAAGCTGGGTTCTCACTGCA
CTCATCTCTGTTGT-3⬘]). As previously described (16), the PCR product of
the target gene was directionally cloned into the donor vector pDONR207
using the BP Clonase-mediated recombination reaction (Invitrogen) to create
an entry vector. The entry vector was then subjected to an LR Clonasemediated recombination reaction (Invitrogen) to introduce two copies of the
target gene into the destination vector pFANTAi4 to make a GFP-bys1 gene
RNAi vector (pFi-bys1) (16).
Transformation of B. dermatitidis. Agrobacterium-mediated gene transfer was
used in all transformations as previously described (23). Transformants were
selected on 3 M agar supplemented with 100 ␮g/ml hygromycin B (A. G. Scientific Inc., San Diego, CA) and 200 ␮M cefotaxime (Sigma-Aldrich).
Silencing of B. dermatitidis yeast-phase-specific gene BYS1. Strains 26199-GFP
and 14081-GFP were transformed with control vectors pH2AB-GFPi, containing
an inverted repeat of GFP sequences (GFP-only RNAi), and pBTS4, lacking the
RNAi hairpin cassette (non-RNAi control), or with GFP-bys1 RNAi vectors.
RNAi candidate transformants were randomly selected, transferred to 3 M agar
supplemented with 100 ␮g/ml hygromycin B, incubated at 37°C, and screened for
GFP using a fluorescence detector (FlourImager SI, Vistra Fluorescence, or
Versadoc; Bio-Rad, Hercules, CA). Selected colonies were further examined by
bright-field and fluorescence microscopy (BX60; Olympus), and photographic
images were obtained using a digital camera (DEI-750; Optronics Engineering).
Fluorescence detection of BAD1 and ␣-(1,3)-glucan. For flow cytometry, yeast
cells were stained with anti-BAD1 monoclonal antibody DD5-CB4 (15) (1:50) at
37°C for 30 min and washed once with phosphate-buffered saline (PBS)–1%
bovine serum albumin (BSA). Goat anti-mouse antibody conjugated with phycoerythrin (1:50) was added to the yeast cell suspension, incubated at room
temperature for 30 min, and washed with PBS–1% BSA. The cells were fixed
with 2% paraformaldehyde for 30 min before GFP and BAD1 measurement by
flow cytometry. Microscopic cell surface detection of ␣-(1,3)-glucan was done as
described previously (11).
Northern blot analysis. RNA blot hybridization used standard protocols (8).
Formaldehyde-agarose gels for Northern blot analysis were loaded with 15 ␮g of
total RNA per lane. The probe for B. dermatitidis GAPDH was a 0.75-kb EcoRI
fragment from pCR2.1-GAPDH#1 (provided by Laurens Smith, Idaho State
University). A 421-bp fragment probe for B. dermatitidis BYS1 was amplified by
PCR from pFi-bys1 (the GFP-bys1 RNAi vector used in this study) using primers
BdBys1R (5⬘-GCCACTGCTTACGCCTGTC-3⬘) and BdBys1F (5⬘-TCTCACT
GCACTCATCTCTGTTGT-3⬘). Fragments for probes were gel purified using a
QIAquick gel extraction kit (Qiagen) and radiolabeled with [32P]dCTP using a
Prime-a-Gene probe-labeling kit (Promega, Madison, WI). Radioactive signals
from probed and washed membranes were detected using a PhosphorImager
2523
(Storm860; Molecular Dynamics, Sunnyvale, CA). The PhosphorImager signal
levels were quantified using Quantity One Software (version 4.6.8) from Bio-Rad
Laboratories.
Assessment of virulence in experimental pulmonary infection. C57BL6 mice
were infected with either yeast cells from strain 26199 or spores from strain
14081 of three independent isogenic GFP-bys1 cosilenced isolates or controls
(including the GFP sentinel strains without any RNAi and the sentinel strain that
had been transformed with either the non-RNAi vector or the GFP-only RNAi
vector). Mice (n ⫽ 8 to 10/group) received 500 yeast cells or spores intratracheally. Signs of disease during daily monitoring, survival over 14 days, and burden
of infection (CFU count) in harvested lungs at day 14 after infection were
analyzed. Statistically significant differences between CFU counts were determined using the Wilcoxon rank test for nonparametric data (10).
Fungal cell morphology in infected lungs. Sections from paraffin-embedded,
formalin-fixed lungs from up to 10 different infected mice for each strain tested
were stained with hematoxylin-eosin and Gomori’s methenamine silver stain.
Organisms were counted and evaluated for yeast or pseudohyphal morphology at
a magnification of ⫻400 on a monitor after digital photography by a trained
pathologist who was blinded to the identity of the samples. Counts were evaluated in 10 random fields of 0.67 mm2 and expressed as percent pseudohyphae (of
the total count per field). The significance of differences between experimental
and control groups was based on logistic regression models.
RESULTS
BYS1 and BYS1-related genes. Analysis of the deduced
amino acid sequence of BYS1 revealed a putative signal peptide, indicating that the protein may be targeted to the endoplasmic reticulum-Golgi apparatus and possibly secreted. To
see if homologues with known activities or sequence motifs
could help to predict BYS1 gene function, BLAST analyses
were performed using the NCBI database or the Broad Institute Fungal Genome Initiative (http://www.broadinstitute.org
/science/projects/fungal-genome-initiative/). From these analyses, a possible paralog of BYS1 with only 34% amino acid
identity (expected value ⫽ 9e⫺14) was identified in the B.
dermatitidis genome. In addition, a BYS1 superfamily conserved domain (cl04669) was identified in the Conserved Domain Database. Within this superfamily are members of other
fungal species, including the closely related species H. capsulatum and Coccidioides spp., but the identified sequences encode proteins more closely related to the BYS1 paralog (by
both amino acid identity and reciprocal BLAST) than to BYS1
itself. None of these predicted proteins has known functions
or conserved protein motifs, other than being placed in the
BYS1 superfamily (9; data not shown). Therefore, from the
BYS1 sequence information alone, it is not possible to predict BYS1 gene function.
Identification of bys1-silenced strains. As a first step in the
functional analysis of BYS1, we sought to generate strains with
reduced BYS1 expression and determine if they displayed altered morphological or pathogenic properties. The GFP sentinel RNAi tracking strategy was used to rapidly identify B.
dermatitidis bys1-silenced strains (16). We constructed an
RNAi plasmid for BYS1 by using a 421-bp fragment of the B.
dermatitidis BYS1 coding sequence from the cDNA of strain
26199. The 421-bp BYS1 sequence was cloned into the GFP
sentinel RNAi vector pFANTAi4 (16) to create the GFP-bys1
RNAi vector (pFi-bys1). The B. dermatitidis 26199-GFP reporter strain was then transformed with pFi-bys1. We screened
GFP-bys1 RNAi transformants for a low GFP signal level as a
sentinel of reduced BYS1 expression. Thirty-two clones of
GFP-bys1 RNAi were randomly selected for GFP signal quantification (Fig. 1A). As controls, eight transformants each from
DOWNLOADED FROM iai.ASM.ORG - at Univ of Wisconsin - Mad July 15, 2010
VOL. 78, 2010
DOWNLOADED FROM iai.ASM.ORG - at Univ of Wisconsin - Mad July 15, 2010
FIG. 1. Silencing GFP-bys1 in B. dermatitidis GFP sentinel strains. (A) GFP signal levels of individual colonies of 8 each of the control
transformants (non-RNAi and GFP-only RNAi) and 32 randomly selected GFP-bys1 RNAi transformants detected by FluorImager. (B) The GFP
signal level of cells from individual colonies was measured by flow cytometry, and the mean fluorescence intensities are shown. Control strains:
non-RNAi, n ⫽ 2; GFP-only RNAi, n ⫽ 2. GFP-bys1 RNAi strains, n ⫽ 32. The number designation for each of the GFP-bys1 RNAi strains is
indicated. (C) Northern blot analysis to confirm BYS1 silencing in GFP-bys1 RNAi strains. Each lane represents 15 ␮g of total RNA. The same
blot was probed for BYS1, exposed to the Phosphorimager screen, stripped, and reprobed with GAPDH (see Materials and Methods). Shown is
a composite image of the two probes, with only the specific hybridizing regions shown for each. The relative BYS1 expression of each strain is
indicated below and was obtained by dividing the BYS1 signal level by the GAPDH signal level (to correct for loading differences). GFP-expressing
strain 26199-GFP, two control strains (non-RNAi and GFP-only RNAi), and two GFP-bys1 RNAi strains are shown.
2524
B. DERMATITIDIS BYS1
2525
FIG. 2. Rough colony phenotype correlates with sentinel GFP silencing. 7H10 slants were inoculated with the indicated strains and incubated
at 37°C for 3 weeks. The mean fluorescence intensity from the flow cytometry analyses in Fig. 1B is shown below each image. There is a strong
correlation between a low GFP value and a rough colony appearance. Note that for GFP-bys1 RNAi strain 27, silencing failed and the colony
phenotype is smooth.
the GFP reporter (26199-GFP) transformed with a non-RNAi
or a GFP-only RNAi vector were also randomly selected (Fig.
1A). Non-RNAi transformants showed GFP signal levels as
high as that of the reporter strain 26199-GFP, while GFP-only
RNAi transformants showed low GFP signal levels equivalent
to that of untransformed wild-type strain 26199 (data not
shown). GFP-bys1 RNAi transformants showed a wide range
of GFP signal levels (Fig. 1B). Compared to the non-RNAi
control, about 50% of the GFP-bys1 transformants showed an
⬃10-fold GFP signal level reduction (Fig. 1B). We analyzed
these low-GFP transformants for mRNA abundance and phenotypic alterations.
Northern analysis of BYS1 transcript. To prove that the
sentinel reporter and the phenotypes accurately signaled BYS1
silencing, we determined relative mRNA levels by Northern
blot analysis for representative transformants of wild-type
26199, non-RNAi control, GFP-only RNAi control, and GFPbys1 RNAi lines. The blotted membrane was probed sequentially with BYS1 and GAPDH probes (Fig. 1C). The wild type,
non-RNAi, and GFP-only RNAi controls had a strong BYS1
signal. The BYS1 transcript was sharply reduced in the GFPbys1 RNAi transformants to nearly background levels (Fig.
1C). The GAPDH transcript signal level was used to correct
for RNA loading differences, and the BYS1 mRNA levels in
the silenced strains were found to be reduced to less than 3%
of the control levels (Fig. 1C).
bys1-silenced strains exhibit a defect in morphogenesis.
Changes in yeast cell surface components (11, 14) or cell morphology (i.e., pseudohyphae [17]) often result in a macroscopic
change from a smooth-colony phenotype to a rough colony
surface. GFP-bys1 RNAi transformants with a low GFP signal
level showed a rough-colony phenotype on 7H10 agar medium
that was not apparent after the initial passage of the cells but
appeared as the cultures were incubated beyond 2 weeks. In
contrast, representative GFP-bys1 RNAi strains with a normal
GFP signal level, non-RNAi controls (normal GFP signal
level), and GFP-only controls (low GFP signal level) all retained smooth colony surfaces during early and extended culture, as expected for normal yeast cells (Fig. 2). Microscopically, the GFP-bys1 RNAi strains with a low GFP signal level
and a rough colony appearance also exhibited pseudohyphal
morphology, whereas the controls strains, including the nonRNAi (normal GFP signal level) and GFP-only RNAi (low
GFP signal level), demonstrated a homogeneous, round budding-yeast appearance (Fig. 3). Despite the underlying defect
in the GFP-bys1 RNAi strains, there was no effect on the
growth rate, as these strains (and the control strains) grew at
the same rate as wild-type cells based on optical density measurements of liquid cultures. Growth rates were measured for
freshly subcultured strains in early- to mid-log-phase liquid
cultures. Under these conditions, both the bys1-silenced and
control stains exhibit budding-yeast morphology (data not
shown). The cells grown in liquid culture did not adopt a
pseudohyphal morphology during short-term growth. The
growth rates of pseudohyphal-form cells after extended culture
were not quantified.
Measurements of cell surface and cell wall intactness. Because of the changes in colony appearance and morphology
resulting from BYS1 silencing, we sought to probe for perturbations of yeast cell wall content and chemical sensitivity that
may be associated with these altered morphologies. Alteration
of the cell wall ␣-1,3-glucan content of B. dermatitidis grown at
37°C results in a rough colony appearance (11). Therefore, the
GFP-bys1 RNAi strains were tested for loss of ␣-1,3-glucan on
FIG. 3. Propensity for pseudohyphal growth at 37°C with RNAi
silencing of BYS1. Cells were removed from 7H10 slants grown at 37°C
for 3 weeks and viewed by fluorescence (left) or bright-field (right)
microscopy. Strain names are indicated on the far left. Controls: nonRNAi and GFP-only RNAi. GFP-bys1 RNAi: strains 29 and 35. Note
that the GFP-bys1 RNAi strains (29 and 35) have low GFP fluorescence, as well as many abnormally elongated cells, compared to the
typical budding yeast cells of the control strains.
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VOL. 78, 2010
KRAJAEJUN ET AL.
the cell surface using a fluorescent-antibody microscopic assay
(11). There was no difference in the intensity of fluorescence
detected in the GFP-bys1-silenced strains compared to that of
the controls (data not shown). Immunostaining and flow cytometry were used to determine if the morphological effect of
BYS1 silencing alters the localization of the yeast-phase-specific virulence factor BAD1 on the cell surface. As expected,
BAD1 knockout strain 55 (n ⫽ 1) lacked surface BAD1 (and
GFP), whereas the non-RNAi controls (n ⫽ 8) expressed surface BAD1 and GFP and the GFP-only RNAi controls (n ⫽ 8)
expressed surface BAD1 but had reduced GFP. For the GFPbys1 strains, the results were similar to those of the GFP-only
RNAi control (n ⫽ 32), indicating no alteration in the expression of surface BAD1 (data not shown).
Some mutant strains of fungi with altered cell walls are more
sensitive than their wild-type progenitors to the inhibiting effect of the cell wall-binding compounds Congo Red and Calcofluor White (17). Therefore, we tested whether a bys1 RNAi
strain had increased growth sensitivity to these compounds by
culturing them in liquid HMM supplemented with increasing
amounts of either inhibitor (0.2 to 20 ␮g/ml). We did not find
increased sensitivity of the bys1 RNAi strain for either Congo
Red or Calcofluor White compared to the GFP-only RNAi
control (data not shown).
Experimental infection of bys1-silenced strains in mice. As a
test for altered virulence in the GFP-bys1 RNAi strains, we
used a murine model of infection (26). For experimental infection, inocula of 500 cells of the 26199-GFP reporter, a
representative non-RNAi transformant, a representative GFPRNAi transformant, and three independent GFP-bys1 RNAi
strains with the lowest GFP signal level, all grown at 37°C, were
introduced intratracheally into mice (8 to 10/group). In order
to avoid differences in the efficiency of intratracheal infection
using morphologically different cells, we used cells from fresh
cultures for the silenced and control strains that were similar in
yeast morphology. To investigate whether the decreased BYS1
expression impairs initial lung colonization, 2 or 3 mice from
each group were sacrificed for lung CFU counting at day 1
postinfection. The average lung CFU counts of the groups
were as follows: 26199-GFP reporter, 716 (n ⫽ 3); non-RNAi
transformant, 854 (n ⫽ 2); GFP-RNAi transformant, 894 (n ⫽
2); the 3 GFP-bys1 transformants, 216 (n ⫽ 3), 334 (n ⫽ 2), and
683 (n ⫽ 3). The rest of the mice were monitored for the onset
of disease symptoms, which appeared at similar times in all of
the groups. At 14 days, all of the animals in all of the groups
appeared moribund and some of the mice in both the control
and bys1 RNAi groups had died. At this time, lungs were
harvested and the burdens of infection were determined (Fig.
4A). In all of the groups, the mice had a burden of infection in
the range of 106 CFU. None of the GFP-bys1 RNAi strains
were significantly different (P ⬍ 0.05) from the GFP-only
RNAi strain (Fig. 4). Analysis of the yeast colonies obtained
from mouse lungs indicated that GFP expression was still silenced in the RNAi strains (data not shown). These results
indicate that silencing induced by bys1 RNAi did not lead to
decreased virulence.
BYS1 does not regulate phase transition or impair conidiation. To extend the analysis of the effects of BYS1 silencing to
phase conversion and conidiation, we generated a second set of
bys1 RNAi lines in B. dermatitidis strain 14081, since this strain
INFECT. IMMUN.
FIG. 4. Silenced bys1 mutant strains have normal virulence. Mice
(8 to 10 per group) were inoculated intratracheally with 500 cells of
control or silenced bys1 mutant strains. At 14 (A) or 21 (B) days after
infection, the mice were moribund and were sacrificed to determine
the burden of infection in the lungs. The resulting CFU counts per
lung are plotted for each strain. The data are presented as box plots in
which the top and bottom of the box represent the 75th and 25th
percentiles, the median is indicated by the horizontal line, and the top
and bottom of the vertical line indicate the maximum and minimum
values for each group of mice. (A) Yeast cells, 26199 genetic background. 26199 GFP, 26199 expressing the GFP reporter from the
BAD1 gene promoter; non-RNAi, transformed with vector lacking
RNAi hairpin; GFP-only RNAi, transformed by tDNA with a GFPonly hairpin; bys1 RNAi strains 29, 32, and 35, independent transformants with GFP-bys1 RNAi. Although bys1 RNAi strain 32 had a
significantly lower CFU count than strains 29 and 35 (P ⬍ 0.005), none
of the GFP-bys1 strains were significantly different from the control
strain with GFP-only RNAi (P ⬍ 0.05). (B) Conidia, 14081 genetic
background. Plot labels as in panel A, except that the strains were
generated in strain 14081. bys1 RNAi strain 10 had a significantly
higher CFU count than other bys1 RNAi strains or any of the control
strains (P ⬍ 0.005). bys1 RNAi strains 20 and 36 were not significantly
different from any of the control strains (P ⬍ 0.05).
produces more abundant conidia than strain 26199. Both the
26199 and 14081 GFP-bys1-silenced strains converted to mycelia following a temperature shift from 37°C to 22°C on 3 M
agar. Analysis using light microscopy revealed normal mycelial
morphology and conidiation. Conidia harvested from 14081
GFP-bys1-silenced strains and controls were enumerated, and
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B. DERMATITIDIS BYS1
dilutions were plated on 3 M agar and incubated at 37°C to
determine if the GFP-bys1-silenced strains were defective in
spore germination or conversion back to the yeast form. Based
on colony counts, the silenced and control strains germinated
and gave rise to yeast colonies with the same efficiency in vitro
(data not shown).
To investigate if bys1 RNAi affects morphological conversion from spores to yeast and virulence in vivo, the murine
model of pulmonary infection (above) was used. Here,
C57BL6 mice were each infected intratracheally with an inoculum of 500 conidia (instead of yeast cells) with the 14081
control and GFP-bys1 RNAi strains. Three weeks postinfection, mice infected with the control and GFP-bys1-silenced
strains appeared moribund. All of the GFP-bys1-silenced
strains had lung CFU levels similar to or, in the case of bys1
RNAi strain 10, higher than those of the controls (Fig. 4B).
Colonies that emerged from lung cultures were analyzed for
GFP fluorescence (a total of 50 colonies, from up to 10 lungs
per strain). In the three bys1 RNAi strains, 10, 20, and 36, there
were 3.3-, 7-, and 6-fold reductions in fluorescence, respectively, compared to non-RNAi control strains, demonstrating
that the bys1 RNAi strains maintained silencing in the lungs.
At the time mice were analyzed for the burden of lung
infection, a portion of lung tissue was resected and fixed in
formaldehyde for microscopic analysis. The lung sections were
stained, and the fungal cells were assessed for yeast or
pseudohyphal morphology. For the 14081-GFP and non-RNAi
control strains, pseudohyphal cells were rarely observed
(⬍0.6%). In the bys1-silenced strains, there was an increase in
pseudohyphal morphology up to an average of 4.2% of the
cells of bys1 RNAi strain 36 (individual counts from mouse
lungs ranged from 0% to 7%, with a mean of 3.2%, averaged
from 30 microscopic fields analyzed, representing three different bys1 RNAi strains and a total of 1,541 fungal cells
counted). The proportion of pseudohyphal cells of the bys1silenced-strains in vivo was significantly higher than that in the
control groups (P ⬍ 0.004).
DISCUSSION
Previous work has shown that BYS1 mRNA is expressed at
high levels in yeast cells but rapidly diminishes and disappears
as yeast-to-mycelial conversion occurs following a temperature
shift to 25°C (4, 9). Incubation of mycelia at 37°C leads to the
reappearance of BYS1 within 12 h. BYS1 is postulated to encode an 18.6-kDa protein that contains multiple putative phosphorylation sites, two 34-amino-acid domains with similarly
spaced 9-amino-acid degenerative repeating motifs, and a hydrophobic N terminus, which may act as a signal sequence for
endoplasmic reticulum-Golgi apparatus localization and secretion (9). BLAST analyses of the predicted BYS1-encoded
amino acid sequence against the NCBI and Broad Institute
databases revealed that several fungal species have weakly
matching hypothetical proteins. The identified sequences have
higher identity to a paralog of BYS1 than to BYS1 itself. This
was true even for closely related species, including H. capsulatum, P. brasiliensis, and C. immitis. While there is no predicted function for either BYS1 or the BYS1 paralogs, these
analyses suggest that BYS1 has diverged in sequence from the
others and therefore may also have diverged in function.
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Gene silencing has become a useful tool for investigating gene
function in eukaryotes. However, one limitation to RNAi is the
range, from very low to very high, of the efficiency of silencing of
a target gene among sampled RNAi transformants. Therefore, it
is necessary to monitor the efficiency of gene silencing by measuring decreases in target gene mRNA in individual transformants by a time-consuming and labor-invasive assay such as realtime PCR or Northern blot analysis. Recently, a GFP sentinel
RNAi system was developed to allow easy and rapid screening for
the most efficiently silenced transformants (16, 18). The GFP
signal level is an indicator of the level of silencing in transformants, and the level of GFP fluorescence accurately reports the
level of target gene silencing (16).
The GFP sentinel RNAi system was applied for loss-offunction analysis of BYS1 by transforming a GFP reporter
strain using an A. tumefaciens binary plasmid engineered to
express hairpin RNA containing both GFP and BYS1. Thirtytwo clones of GFP-bys1 RNAi were picked for further analysis.
In these, the degree of GFP silencing varied, and about half of
the transformants had an at least 10-fold reduction in GFP
fluorescence (Fig. 1A and B). RNA blot hybridization showed
nearly background levels of BYS1 mRNA transcript in GFPbys1 RNAi strains with low GFP intensity. In contrast, the
control strains maintained close-to-wild-type levels of BYS1
transcripts (Fig. 1C). Several of the GFP-bys1 RNAi strains
(with low GFP intensity) were analyzed for phenotypes that
would affect the pathogenic life cycle of this organism.
One striking phenotype observed in bys1-silenced strains was
the rough appearance of colonies grown on agar surfaces. This
phenotype is not apparent immediately but develops as the
cultures mature. Microscopically, GFP-bys1-silenced strains at
this stage showed an increased proportion of pseudohyphal
types. Since B. dermatitidis can develop pseudohyphal morphology when grown under stressful conditions (such as hyperosmotic medium; data not shown), we felt it essential to show
that the altered morphology of GFP-bys1 RNAi strains is due
to the silencing of BYS1 and not to a general stress response.
We took great care in generating control strains with which
to compare cell morphology and culture surface phenotypes.
At the same time as for the GFP-bys1 RNAi strains, several
types of control strains were produced by Agrobacterium-mediated transformation. These included strains in which a nonRNAi vector was introduced, and strains harboring an introduced RNAi vector that silences only GFP. These strains were
grown in parallel with the GFP-bys1 RNAi strains, and none of
them had the morphological phenotypes found by silencing
BYS1. Several independently generated bys1-silenced strains
were analyzed (Fig. 2, strains 29, 32, 35, and 47), as was a strain
that failed at silencing BYS1 (Fig. 2, strain 27). The strains with
strong GFP-bys1 silencing all show the rough surface phenotype, and the strain with weak GFP-bys1 silencing had morphological and surface characteristics of wild-type B. dermatitidis. Following the infection of mice, bys1-silenced cells had a
mild but significant increase in the proportion of pseudohyphal
cells in the lungs of these animals. The visualization of
pseudohyphal forms of B. dermatitidis in vivo is remarkable in
itself, which is underscored by their absence in the control
strains used to infect mice.
These phenotypes suggest that BYS1 may play a role in cell
wall formation or morphogenesis in B. dermatitidis. Since the
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KRAJAEJUN ET AL.
BYS1 gene sequence appears to encode a signal peptide at its
amino terminus, it is enticing to speculate that the BYS1-encoded
protein is secreted and localized on the cell surface, where it may
impact cell surface structure and perhaps overall cell morphology.
Since the bys1-silencing phenotype only develops as the cells mature, it may be that the loss of BYS1 affects the cell wall in a way
that is conditional on cell density, nutrient availability, or another
property inherent to older cultures.
BAD1 is a surface protein of B. dermatitidis that is an essential virulence factor whose distribution on the yeast cell
surface can be affected in strains with altered yeast cell morphology and surface structure (11, 17). Therefore, we tested
whether bys1 silencing results in altered BAD1 distribution on
the cell surface. However, this is not the case, as immunological detection of BAD1 on the surface of bys1-silenced strains
was similar to that on control cells (data not shown).
Several types of experimental results point to connections
between the yeast cell surface, cell morphology, and virulence.
These include the need for BAD1 on the surface for virulence
but also observations that changes in cell wall glucan composition can decrease virulence (11) and that deletion of DRK1,
which results in a failure to form yeast cells at 37°C, also
ablates virulence (17). These published findings suggest the
possibility that bys1-silenced strains, with their conditional
morphological defect, could also have an effect on virulence.
However, we found no significant reduction in virulence in
bys1-silenced strains compared to the controls. As the proportion of pseudohyphal cells in the lungs of mice infected with
the bys1-silenced strains only amounted to a small fraction of
the total fungal cells, we cannot formally exclude the possibility
that the bys1-silenced cells with a pseudohyphal phenotype
have altered virulence compared to those with budding yeast
morphology. In contrast to other well-characterized yeastphase-specific genes (BAD1, CBP1, YPS3, and SOWgp) which
are essential for virulence but not morphogenesis, the yeastphase-specific BYS1 gene of B. dermatitidis has a role in morphogenesis but not virulence.
We have provided additional evidence that the GFP sentinel
RNAi system (16) is useful for quickly identifying phenotypes
after disruption of the expression of a target gene in the dimorphic fungus B. dermatitidis. As more whole-genome sequences of dimorphic fungi become available, this will be a
valuable tool in determining which genes may have a role in
dimorphism and virulence.
ACKNOWLEDGMENTS
We thank Laurens Smith, Idaho State University, for the GAPDH
plasmid and Robert Gordon for help with preparation of the figures.
Keegan D. Korthauer and Mike Evans provided assistance with statistical analyses.
This work was supported by grants from the Ellison Foundation
(T.K.) and the USPHS (B.S.K.).
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