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Infection and Immunity, July 1999, p. 3649-3652, Vol. 67, No. 7
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Invasive Lesions Containing Filamentous Forms
Produced by a Candida albicans Mutant That Is Defective in
Filamentous Growth in Culture
Perry J.
Riggle,1
Karl A.
Andrutis,2
Xi
Chen,1
Saul R.
Tzipori,2 and
Carol A.
Kumamoto1,*
Department of Molecular Biology and
Microbiology, Tufts University School of Medicine, Boston,
Massachusetts 02111,1 and Division of
Infectious Diseases, Tufts University School of Veterinary
Medicine, North Grafton, Massachusetts
015362
Received 2 February 1999/Returned for modification 21 March
1999/Accepted 14 April 1999
 |
ABSTRACT |
A Candida albicans efg1 cph1 double mutant is
nonfilamentous under standard laboratory conditions and avirulent in
mice. However, this mutant produced filaments in the tongues of
immunosuppressed gnotobiotic piglets and when embedded in agar,
demonstrating that an Efg1p- and Cph1p-independent pathway for
promotion of filamentous growth exists.
| |
TEXT |
The fungal pathogen Candida
albicans is an important nosocomial pathogen that causes diseases
ranging from superficial mucosal infections to life-threatening
systemic disease. Like primary fungal pathogens, C. albicans
is dimorphic and is capable of growth in a yeast form or in filamentous
forms. Filamentous growth is believed to be an important step in the
invasion-and-pathogenesis process (4, 10).
A variety of stimuli promote filamentous growth in culture. In
particular, growth at 37°C or higher temperatures in media containing
serum or a combination of amino acids promotes hyphal growth (for a
review, see reference 6). Several gene products are
involved in the regulation of filamentous growth, including the
products of the CPH1 (9), EFG1
(12), CZF1 (3), TUP1 (2), and RBF1 (7) genes.
A mutant strain lacking the transcription factors Efg1p and Cph1p is
extremely defective in filamentous growth at 37°C (10). In
this study, laboratory growth conditions that differ from standard conditions for the stimulation of hyphal growth were used to promote filamentous growth and the efg1 cph1 double null mutant was
observed to form filaments. In addition, an alternative animal model,
the immunosuppressed gnotobiotic (IGB) newborn piglet, was used to study the virulence of this mutant. IGB animals were highly susceptible to wild-type C. albicans, and mucosal invasion and
dissemination were consistently observed (1). The efg1
cph1 double null mutant produced lesions containing filamentous
forms in the tongues of IGB piglets. Therefore, these results
demonstrate that an Efg1p- and Cph1p-independent pathway for promotion
of filamentous growth exists in C. albicans and that this
pathway can be utilized during in vivo infection.
Filamentous growth of an efg1 cph1 double null mutant
during experimental infection.
Oral inoculation of newborn
germfree piglets (11) with wild-type C. albicans
SC5314 (5), followed by immunosuppression with cyclosporine
and methylprednisolone (8), resulted in colonization of the
gastrointestinal tract by C. albicans, dissemination to internal organs, and development of oral thrush, conjunctivitis, and
corneal lesions (1). Tongues of animals inoculated with SC5314 were completely covered with fungal material, and culture of
tongue homogenates yielded a mean of 9 × 106 CFU/g
(wet weight) of tissue. To study filamentous growth during this
experimental infection, strain CKY138 (CAI-4
efg1::hisG/efg1::hisG cph1::hisG/cph1::hisG ura3/ura3
ade2::pDBI52), derived from SC5314, was inoculated
orally into germfree piglets. CKY138 was constructed by transformation
of strain can36 (efg1/efg1 cph1/cph1 ura3/ura3; kind gift of
G. Fink) with vector pDBI52 (Ura+) (3). Despite
the reduced virulence of strains lacking Efg1p and Cph1p in mice
(10) and IGB piglets (1), oral inoculation of IGB
piglets led to extensive colonization of the gastrointestinal tract and
development of sparse but clinically apparent, mild thrush lesions and
superficial lesions of the eye. Consistent with the reduced virulence
of the mutant strain, these oral lesions required a much longer time to
develop than did lesions caused by wild-type C. albicans.
Homogenates of tongue tissue containing lesions yielded a mean of
2 × 105 CFU/g (wet weight) of tissue.
To determine the morphology of the organisms in tongue lesions,
infected tissue was fixed in 10% formalin and sectioned.
Immunohistological tests using antibodies produced by repeated
intramuscular injection of formalin-killed C. albicans were
performed. The wild-type organism (SC5314) produced locally invasive
lesions with extensive filamentous growth throughout the keratin and
epithelial layers (Fig. 1A). In some
lesions, organisms were observed in subepithelial layers invading
muscle tissue. The mutant CKY138 (efg1/efg1 cph1/cph1) produced superficial lesions limited to the epithelial layer, but
filamentous growth was clearly observed (Fig. 1B), despite the fact
that this strain has been reported to be nonfilamentous in culture
(10).
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FIG. 1.
Filamentous growth of C. albicans during
experimental infection. Newborn, germfree piglets were inoculated
orally with either SC5314 (wild type) or CKY138 (efg1/efg1
cph1/cph1). Sections of fixed tongue tissue were examined by
immunohistochemical analysis using anti-C. albicans antibody
and photographed at a magnification of ×40. Panels A, tongue section
from an animal inoculated with SC5314; B, tongue section from an animal
inoculated with the CKY138 mutant.
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To demonstrate that the reduced virulence observed with strain CKY138
was due to the
efg1 cph1 mutations, strain HLC84
(
efg1::hisG/efg1::hisG cph1::hisG/cph1::hisG ura3/ura3
leu2::EFG1+) was constructed by
transformation of a wild-type copy of the
EFG1 gene from
plasmid HLB134 (
10) into strain can36. This strain
exhibited
nearly normal virulence in the IGB piglet model and
produced lesions in
the tongue that were very similar to wild-type
lesions (
1).
Homogenates of tongue tissue yielded a mean of
6 × 10
6 CFU/g (wet weight) of tissue. Therefore, deletion of
EFG1 and
CPH1 resulted in a mutant strain with
reduced virulence but the
loss of these two transcription factors was
not sufficient to
eliminate filamentous growth during infection or the
development
of invasive lesions in the
tongue.
In the cornea, both SC5314 (wild type) and CKY138 (
efg1/efg1
cph1/cph1) produced lesions as well. In lesions produced by
SC5314,
extensive filamentous growth was observed while lesions
produced
by CKY138 contained predominantly yeast cells (data not
shown).
In addition, an
efg1 cph1 double null strain failed
to become
filamentous when engulfed by macrophages
(
10). Therefore, filamentous
growth of mutant CKY138
was not observed in all tissues, indicating
that different sites
within the host may provide different signals
for initiation of
filamentous
growth.
Laboratory conditions that promote filamentous growth of mutant
CKY138.
Previous results demonstrated that physical environmental
cues due to growth within a matrix promoted filamentous growth of C. albicans in the laboratory (see Fig. 3A) (3).
Plating of cells on a macroscopically rough agar surface was found to
have a similar effect. A rough agar surface was produced by shaking molten YPS agar (1% yeast extract, 2% Bacto Peptone, 2% sucrose, 1%
agar) to introduce bubbles and allowing the agar to solidify. Cells
were then plated on the surface. After incubation at 25°C, colonies
that grew on the smooth parts of the plate developed into colonies
having sharp edges composed of yeast cells (Fig. 2A and
B), while colonies that grew on the rough
parts of the plate developed into flat, ragged colonies that produced
filaments after several days (Fig. 2C and D).
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FIG. 2.
Growth of wild-type cells on the surface of rough or
smooth agar. Cells of SC5314
(EFG1+/EFG1+
CPH1+/CPH1+) were plated
on YPS plates containing rough and smooth areas and incubated at 25°C
(see text). At various times, colonies were observed and photographed
with a Nikon TM300 inverted-phase microscope with a 10× objective. The
arrow in panel C indicates the edge of the colony. The arrow in panel D
indicates filaments. Panels A and B show colonies grown on a smooth
surface (A, 1 day; B, 3 days). Panels C and D show colonies grown on a
rough surface (C, 1 day; D, 3 days).
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Strain CKY138 (
efg1/efg1 cph1/cph1) does not produce hyphae
at 37°C in serum-containing media (
10). This result is
striking
because this condition strongly stimulates hyphal growth of
wild-type
cells. However, CKY138 exhibited filamentous growth in
response
to plating in a matrix (Fig.
3B)
or on a macroscopically rough
surface (Fig.
3C). Wild-type
C. albicans is capable of forming
both hyphae, elongated cells
lacking constrictions at the septa,
and pseudohyphae, variably
elongated cells in chains with constrictions
at the septa. To determine
whether the filaments produced by CKY138
were hyphae or pseudohyphae,
Calcofluor white staining of cells
extracted from colonies was
performed (
3). Constrictions were
observed at septa
separating the cells in filaments, indicating
that the filaments were
pseudohyphae (Fig.
3D).
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FIG. 3.
Filamentous growth of strain CKY138. Exponentially
growing cells of CKY101 (wild type) (A) and CKY138 (efg1/efg1
cph1/cph1) (B) grown in YPD medium (1% yeast extract, 2% Bacto
Peptone, 2% glucose) at 30°C were mixed with molten YPS medium and
plated. After 3 days of incubation at 25°C, colonies were
photographed at a magnification of ×10. Panel C shows exponentially
growing cells of strain CKY138 plated on a rough YPS agar surface,
incubated at 25°C for 5 days, and photographed at a magnification of
×10. Panel D shows CKY138 cells extracted from a colony grown on rough
YPS agar, stained with Calcofluor white, and photographed at a
magnification of ×100 by using a standard
4',6-diamidino-2-phenylindole (DAPI) filter set.
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To compare the responses of wild-type and CKY138 cells to growth
conditions, cells were grown as previously described (
3)
and
embedded or plated on a rough surface. In some experiments,
wild-type
strain CKY101, constructed by transformation of CAI-4
(
ura3/ura3) with vector pDBI52 (
3), was used.
Either 200 embedded
colonies or 50 to 100 colonies growing within rough
areas were
observed at various times. A colony that contained at least
20
filaments protruding from the periphery of the colony was
arbitrarily
defined as a filamentous colony. The kinetics of
filamentous growth
of wild-type strains and the CKY138 mutant were
similar under
both conditions (Fig.
4A and
B). In contrast, cells growing on
the
surfaces of smooth plates did not produce filaments until
6 or 7 days
of incubation (data not shown) (
3).
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FIG. 4.
Kinetics of filamentous growth of strain CKY138 and
wild-type C. albicans. Cells of strains CKY101 (wild type),
CKY138 (efg1/efg1 cph1/cph1
ade2::Ura+), SC5314 (wild type), and CKY169
(czf1/czf1 cph1/cph1 ade2::Ura+) were
embedded in YPS agar (A) or plated on rough YPS agar surfaces and
incubated at 25°C. The percentage of filamentous colonies is plotted
as a function of time. Panels: A, CKY101 and CKY138 embedded; B,
SC5314, CKY138, and CKY169 plated on rough YPS agar.
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Not all
C. albicans strains become filamentous on rough
agar. Mutant strain CKY169 (CAI-4
czf1::hisG/czf1::hisG
cph1::hisG/cph1::hisG ade2::pDBI52),
lacking the putative transcription factors Czf1p
and Cph1p, was
defective in producing hyphae under these conditions
(Fig.
4B) and was
also delayed in producing filaments in response
to matrix embedding
(
3). Therefore, the responses to matrix
embedding and to
growth on a rough surface were affected by mutations
in the same genes,
indicating that the two processes were regulated
similarly.
These experiments and previous studies demonstrate that cues from the
physical environment regulate hyphal growth in
C. albicans.
In these experiments, the rich medium used and the low temperature
did
not promote rapid filamentous growth of
C. albicans when
cells
were growing on the surface of a smooth layer of agar
(
3) or
in a liquid culture (data not shown). Since an
individual plate
contained some rough parts and some smooth parts, the
chemical
environments of colonies growing on rough and smooth parts of
the plate would be expected to be the same, yet these colonies
developed very differently. Therefore,
C. albicans responds
to
the nature of the physical environment and, in some physical
environments,
produces hyphae. The existence of another well-studied
response
to the physical environment, thigmotropism (
6), a
tropism caused
by contact with a solid surface, demonstrates that
C. albicans must possess mechanisms for sensing the physical
environment and
responding to that environment. The question of whether
responses
to growth on a rough surface, embedding and thigmotropism,
use
common sensing mechanisms awaits further
study.
Filamentous growth in the tongues of IGB piglets of a mutant strain,
CKY138 (
efg1/efg1 cph1/cph1), that is deficient in response
to standard chemical and temperature cues for induction of hyphal
growth demonstrates that there is a pathway for regulation of
filamentous growth that is independent of Efg1p and Cph1p and
that this
pathway can function during experimental infection.
Despite its defect
in forming hyphae in response to a temperature
shift and serum, CKY138
responded to growth on a rough surface
or to matrix embedding and
produced pseudohyphae. Since important
pathways for stimulation of
hyphal growth are defective in this
strain, this mutant may have
allowed detection of the effects
of a physical environment sensing
mechanism that stimulates filamentous
growth of
C. albicans
in tissue. Further genetic study of the
physical environment sensing
pathway may reveal genes that are
important for tissue invasion during
oral
infection.
| |
ACKNOWLEDGMENTS |
We thank Ralph Isberg and Andrew Camilli for helpful discussions
and comments on the text. We are also grateful to Gerry Fink for
strains and plasmids and to Angela Giusani for technical assistance. The use of resources and facilities of the Division of Infectious Diseases at the Tufts University School of Veterinary Medicine is
greatly appreciated.
This work was supported in part by National Institute of Allergy and
Infectious Diseases grants R01 AI 38591 (to C.A.K.) and K08 AI01407 (to
K.A.A.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Microbiology, Tufts University, 136 Harrison
Ave., Boston, MA 02111. Phone: (617) 636-0404. Fax: (617) 636-0337. E-mail: ckumamot{at}opal.tufts.edu.
Editor:
T. R. Kozel
| |
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Infection and Immunity, July 1999, p. 3649-3652, Vol. 67, No. 7
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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