From the Department of Stomatology, University of
California at San Francisco, California 94143-0422 and
Merck
Research Laboratories, Rahway, New Jersey 07065-0900
Received for publication, September 21, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Deletion of the kexin gene (KEX2) in
Candida albicans has a pleiotropic effect on phenotype and
virulence due partly to a defect in the expression of two major
virulence factors: the secretion of active aspartyl proteinases and the
formation of hyphae. kex2/kex2 mutants are
highly attenuated in a mouse systemic infection model and persist
within cultured macrophages for at least 24 h without causing
damage. Pathology is modest, with little disruption of kidney matrix.
The infecting mutant cells are largely confined to glomeruli, and are
aberrant in morphology. The complex phenotype of the deletion mutants
reflects a role for kexin in a wide range of cellular processes. Taking
advantage of the specificity of Kex2p cleavage, an algorithm we
developed to scan the 9168 open reading frames in Assembly 6 of the
C. albicans genome identified 147 potential substrates of
Kex2p. These include all previously identified substrates, including
eight secreted aspartyl proteinases, the exoglucanase Xog1p, the
immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative
Kex2p substrates identified include several adhesins, cell wall
proteins, and hydrolases previously not implicated in
pathogenesis. Kexins also process fungal mating pheromones; a
modification of the algorithm identified a putative mating pheromone
with structural similarities to Saccharomyces cerevisiae
Serine proteinases of the kexin/subtilisin superfamily activate
precursor forms of many exported eukaryotic proteins by specific endoproteolytic cleavage adjacent and C-terminal to dibasic residues. Members of the superfamily play a crucial role in a large and varied
set of biological processes in animals, plants, and fungi. In metazoan
cells, kexins participate in both the constitutive and regulated
secretory pathways, with substrates including hormones, serum proteins,
neuropeptides, cell surface receptors, components of the extracellular
matrix (ECM),1 and
proteinases, which modify the ECM (1). Furin, a member of the mammalian
kexin family, processes several ECM metalloproteinases requisite for
the dissemination of cancer cells (2). Kexins have also been suborned
to mediate the maturation of viral proteins and bacterial toxins (3).
In fungi, kexins cycle between the outer face of the Golgi and the
prevacuolar compartment where they process proteins involved in
maintenance and remodeling of the cell wall (4, 5), proteins associated
with the formation of aerial hyphae (6), The Kex2p protein of Candida albicans, an opportunistic
fungal pathogen, participates in pathways that lead to the expression of two of the organism's best characterized virulence factors: hyphal
formation and the secretion of proteinases. Under hyphal-inducing conditions, kex2/kex2 cells are larger than
KEX2/KEX2 yeast, often have multiple buds and
nuclei, and form short, thick, stubby protrusions instead of normal
hyphae (8). C. albicans kex2/kex2 mutants aberrantly process a secreted aspartyl proteinase (SAP), leading to
diminished secretion of the enzyme (8). In this study, we show that
kex2/kex2 C. albicans strains are markedly
attenuated in a mouse model of systemic infection. In systemic
candidiasis, where the primary site of pathogenesis is the kidney, the
mutants are significantly less able to invade the kidney matrix than
the wild type. Furthermore, the null strains do not produce hyphae either when ingested by macrophages, when grown embedded in agar, or
when disseminated in mice.
Because the pleiotropic nature of the null
kex2/kex2 phenotype cannot be entirely explained
on the basis of known Kex2p substrates, we took advantage of the
complete sequencing of the C. albicans genome
(www-sequence.stanford.edu/group/candida/) to develop a purely
informatic approach to identify and characterize potential Kex2p
preproprotein substrates. We identified 147 ORFs whose products are
potentially cleaved by Kex2p. Among these, a number of ORFs encode
hydrolases, adhesins, cell wall components, and outer membrane proteins. In aggregate, the list of candidate Kex2p substrates represents a range of secreted and cell surface-associated activities that are important at the host-fungal interface, some of which are
likely to play key roles in pathogenesis. Additionally, one of the
putative substrates has structural motifs characteristic of S. cerevisiae C. albicans Strains and Growth Conditions--
Strains used in
this study and their relevant phenotypes are: 1) SC5314, a wild-type
blood isolate, 2) CNA1,
Macrophage Strains and Procedures--
Murine macrophage cell
line P388D1 was maintained in Ham's nutrient mixture F12 supplemented
with 100 units/ml penicillin, 100 µg/ml streptomycin, GlutaMAX
(Invitrogen), and 10% fetal calf serum. Macrophages were grown to
confluence in 100 mm2 plastic tissue culture dishes
(~1.2 × 106 cells). C. albicans were
grown for 3 days in YEPD, washed twice with distilled water, suspended
in Ham's F12 without serum, counted, and combined with macrophages at
a multiplicity of infection (moi) of 5.
Mouse Strains and Procedures--
Host neutrophil function is
the most important determinant in the outcome of human systemic
infections (20), and so the virulence of C. albicans was
measured in DBA/2N mice, which are deficient in complement component 5 and impaired in their ability to recruit neutrophils to foci of fungal
growth (21). The DBA/2N mouse model therefore highlights damage due to
direct effects of the fungus while minimizing the contribution from
host inflammatory cells. DBA/2N mice are highly sensitive to fungal
infection; the infective dose 50% (ID50) of C. albicans SC5314 in this strain is lower than that typically
observed in other strains of mice (Table I and Ref. 21).
C. albicans was cultured at 35 °C for 24 h on
Sabouraud dextrose agar (SDA) plates (22). Yeast were scraped from the
surfaces of 1 or 2 plates, suspended in sterile physiological saline,
and enumerated both with a hemocytometer and serial dilution and
plating on SDA. The virulence of each strain was determined in female DBA/2N mice (Taconic Farms, Germantown, NY) inoculated i.v. into the
lateral tail vein with 0.2 ml of a 10-fold titrated suspension of yeast
at doses of 102-107 CFU per mouse (5 mice were
injected at each dose). The ID50 values were estimated at
4, 7, 14, and 21 days after challenge by the method of Knudson and
Curtis (23). Data are presented as the number of yeast in the initial
inoculum that are lethal to half of the animals on the indicated date.
Animals were treated in accordance with the highest standards for the
humane handling, care, and treatment of research animals using
protocols approved by the Merck Institute Institutional Animal Care and
Use Committee. Procedures for the care and use of research animals at
Merck meet or exceed all applicable local, national, and international
laws and regulations (22).
Histopathology--
Kidneys were removed from a separate group
of DBA/2N mice 2 days after infection with 106 wild-type or
kex2/kex2 (CNA3) organisms and placed into
phosphate-buffered saline containing 10% formalin in order to
investigate the histopathology of infection. A high dose of organisms
was necessary to ensure that infected mice did not completely clear
strain CNA3. The tissues were dehydrated, embedded in paraffin, and
sectioned by standard methods. Sections were stained with hematoxylin
and periodic acid-Schiff, then photographed using a Nikon FM2 mounted
on a Zeiss Axioplan fluorescent microscope. Animals used for this part
of the study were maintained under conditions suggested by a
veterinarian and approved by the UCSF Committee on Animal Research.
In Silico Studies--
Assembly 6 of the C. albicans
genome was constructed by the Stanford Genome Technology Center (SGTC)
and the translated ORFs (tORFs, >100 amino acids) are available
(www.sequence.stanford.edu/group/candida/). Self-Blast of Assembly 6 ORFs, to detect alleles and gene families, was performed by S. Scherer
and T. Jones at SGTC. PSORT II (24), source code courtesy of Kenta
Nakai, was used to assign PSG discriminant scores to the tORFs. The PSG
score is calculated from both the hydrophobicity of the central part of
a putative signal sequence and the net charge of its N terminus
(psort.nibb.ac.jp/). Those tORFs with a PSG score of >4 were
considered to be proteins with a leader sequence, but not necessarily
with a signal peptidase cleavage site. Scripts to generate a list of
Kex2p substrates based on PSG value and Kex2p-cleavage site motif, as
well as to detect proteolytic cleavage patterns characteristic of
pheromones, were written in Perl 5.2 and are available upon request.
WU-Blast 2.0 (blast.wustl.edu/) was used to compare the list
to GenPept release 127.0 (www.ncbi.nlm.nih.gov/genbank/), and HMMer 2.2 (hmmer.wustl.edu/) was used to compare the list to Pfam release 7.0 (pfam.wustl.edu/). Both Blast/GenPept and HMMer/Pfam analyses were
performed locally on a Linux 2.2.17 desktop workstation. The resulting
output was parsed with simple in-house Perl scripts, uploaded into a
local relational data base, and annotated manually.
kex2 Mutants Are Highly Attenuated in Systemic Murine
Infections--
The inability of C. albicans
kex2/kex2-null mutants either to process SAPs or to
produce hyphae suggested that these strains might be severely impaired
in pathogenesis and virulence. Therefore we measured the relative
virulence of the KEX2/KEX2 strain SC5314, the
heterozygous KEX2/kex2 strain CNA1, and the
isogenic kex2/kex2 strains CNA3-1 and CNA3-2 in
DBA/2N mice, which are defective in neutrophil responses to fungal
infection (21). The DBA/2N mouse strain is routinely used at Merck for
measuring C. albicans virulence, the ID50 is the
number of yeast cells in the inoculum required to achieve 50%
lethality in test animals at 4, 7, 14, and 21 days post-infection. The
results of such an experiment using the wild-type and kex2
mutant strains are summarized in Table I.
Inactivation of either one or both copies of KEX2 markedly attenuates C. albicans in a gene
dosage-dependent manner. Inocula of ~100 times more CFU
of the kex2/kex2 deletion strains, as compared with the wild type, are required to achieve 50% lethality in mice 4 and 7 days post-infection. At 14-21 days post-infection, 1000 times
more mutant cells are required to achieve lethality equivalent to the
wild type. The heterozygous strain exhibits an intermediate level of
pathogenicity, requiring 15 and 35 times more yeast in the inoculum
than SC5314 did to achieve an ID50 4 and 21 days post-infection, respectively.
kex2 Mutants Invade Kidneys Less Efficiently and Cause Less Damage
than Wild-type Cells--
The most common cause of death in
experimental murine disseminated candidiasis is acute fungal
pyelonephritis due to fungal proliferation and associated damage (21).
Thus the attenuation of kex2/kex2 mutants may
result either because the mutant is more susceptible to clearance from
the bloodstream before reaching the kidneys, or because it has
difficulty crossing the endothelium, or because it is unable to invade
the kidney matrix. To explore these possibilities we infected mice with
either wild-type (SC5314) or kex2/kex2 (CNA3)
C. albicans and inspected the kidneys 2 days post-infection.
The histopathology of kidneys removed from mice infected with SC5314
differs markedly from kidneys isolated from mice infected with CNA3
(Fig. 1). Kidneys of SC5314-infected mice contain multiple foci of proliferating hyphae associated with a general
disruption of the ECM and displacement of host cells (Fig. 1,
A and C). Some of the CNA3 cells concentrate in
foci located in interstitial spaces; these foci are much smaller than those of wild-type cells, often containing fewer than 10 organisms per
nidus (as opposed to hundreds in wild-type infections), and are
associated with little or no morphological damage to the surrounding matrix (Fig. 1B). Overall, we observed far fewer organisms
in the CNA3-colonized kidneys than those in SC5314 infections. CNA3 cells in the kidney fail to form hyphae (Fig. 1D), although
some protuberances are thicker and longer than those of CNA3 observed under any in vitro condition except when embedded in agar
(see below). Unlike SC5314, which forms a broad zone of invasion
throughout the kidney, many of the CNA3 cells remain within the
glomeruli and associated blood vessels, indicating that the mutant is
less able to invade the renal parenchyma than the
KEX2/KEX2 strain. Host inflammatory cells are
absent in areas surrounding CNA3 yeast, suggesting that the attenuation
is not due to cell-mediated killing of the fungi.
KEX2 Is Required for Lysis of Macrophages--
Low numbers of
kex2/kex2 cells in kidneys compared with wild
type can be explained by slower growth or by enhanced clearance by
phagocytic cells. kex2/kex2 yeast grow only
slightly more slowly in various media than the parental strain, and
reach a similar density by stationary phase (data not shown). We
assessed whether the null mutant CNA3 is more susceptible to clearance
by phagocytic cells in vitro. Wild-type C. albicans strain SC5314 is readily phagocytized by macrophage cell
line 388D1, as reported for macrophage cell line IC-21 (25) and
adherent mouse peritoneal macrophages (26). Ingested yeast form hyphae
within 30 min, and by 4 h the hyphae traverse the length of the
macrophage, often extending along the lengths of macrophage processes
(Fig. 2A). The macrophages lyse within 4-6 h, releasing C. albicans hyphae, which bud
new yeast that are subsequently phagocytized by other macrophages. We
did not observe any phagocytosis of hyphae. Within 24 h of exposure to SC5314, the fungi almost completely destroy the macrophage lawn, as evidenced by clearing of the normally opaque macrophage monolayer after removal of medium (data not shown). CNA3 is also phagocytized, but forms aberrant cells with short stumpy projections within the macrophages that are similar to CNA3 cells incubated in
hyphal-inducing liquid media (Fig. 2B); no hyphae are
produced in infected macrophages. After 24 h, small patches of
clearing are seen in the CNA3-inoculated monolayers; however, the lawn of macrophages remains essentially intact (Fig. 2C).
Morphogenesis of Hyphal Forms Requires KEX2--
The inability of
kex2/kex2 mutants to invade the kidney and kill
macrophages can be partially explained by our previous finding that
mutants are defective in both proteinase secretion and hyphal formation
(8). In that study we established that kex2/kex2 mutants are unable to form hyphae in liquid or on solid media (8).
However, subsequent reports have appeared indicating that some
putatively hyphae-deficient mutants are in fact able to form hyphae
when embedded in solid media (19, 25) and in vivo (27). We
therefore asked whether CNA3 was able to form hyphae when embedded in
agar. Within 48 h, SC5314 colonies embedded in solid medium produce filaments consisting largely of hyphae, while CNA3 colonies develop only slightly corrugated surfaces. After 6 days of incubation, SC5314 grows as stellate colonies with hyphal projections extending more than twice the diameter of the main body of the colony, while CNA3
grows as colonies with irregular peripheries that barely extend beyond
the colony mass (Fig. 3, A and
B). Microscopically, the periphery of each CNA3 colony
consists of yeast, pseudohyphae, and aberrant forms similar to those
produced by CNA3 when grown in hyphal-inducing liquid media. The
colonies also contain more elongated aberrantly shaped cells not found
in liquid media but similar to those forms observed in kidney (Fig. 3,
C and D). Approximately 1% of the embedded CNA3
colonies throw out sectors that are clearly more filamentous than other
areas. Cells within the filamentous sectors are predominantly
pseudohyphae, with no true hyphae (Fig. 3E). These sectors
may be products of phenotypic switching which, in this strain, occurs
at a frequency of 1% and manifests as differences in the abundance of
pseudohyphae (28). Alternatively, the sectors may be products of
partial suppression of the kex2 Search for Cleavage Substrates of Kex2p--
It is unlikely that
the absence of Kex2p per se is responsible for the range of
phenotypic defects described above. A more likely explanation is that
Kex2p is required for normal processing and activation of a number of
extracellular proteins that individually or collectively are involved
in defining hyphal morphogenesis, pathogenesis, and virulence. By
"extracellular" we refer to proteins that act wholly or partially
outside of the plasma membrane, including but not limited to cell
surface, cell wall, and secreted proteins. The complete sequencing of
the C. albicans genome, coupled with the highly stereotyped
cleavage recognition motif of ScKex2p in S. cerevisiae,
suggested that we might be able to computationally identify substrates
of Kex2p in C. albicans. To survey the entire C. albicans genome for potential Kex2p substrates, we wrote a computer script which extracts all of the ORFs from Assembly 6 that
encode a protein which: 1) is >100 amino acids in length, 2) possesses
a leader peptide, but not necessarily a signal peptidase cleavage site,
as determined by a modified McGeoh's method (24), and 3) contains a
propeptide < 150 amino acids long, as defined by the motif
P4-P3-Lys-Arg, where P3 can be any
amino acid but P4 cannot be CDFGPS or W. This last
criterion is a weak consensus derived from inspection of known cleavage
substrates of S. cerevisiae ScKex2p (29-31) and from
experimental studies (32, 33). It is possible that C. albicans Kex2p recognizes a cleavage site motif somewhat different
from that of ScKex2p; however, C. albicans KEX2 was isolated
by complementation of a S. cerevisiae Sckex2-null mutant, resulting in the processing of at least two known ScKex2p substrates (8). Based on these criteria, the script extracted 147 candidate Kex2p cleavage substrates from the 9168 predicted proteins
of Assembly 6 of the C. albicans genomic sequence
(www-sequence.stanford.edu/group/ candida/).
Each of the 147 candidate substrates was analyzed by Blast against
Assembly 6, followed by manual inspection of each alignment. This
assessment revealed that 60 candidate ORFs are alleles or fragments of
one another. This reflects the diploid nature of C. albicans
as well as the fragmented nature of Assembly 6, which consists of as
many as 2519 unlinked contigs. Two gene families, with at least three
members each, were identified at this step. One family encodes 9 SAPs,
one of which has not been described in the literature (ORF6.1902), and
the other encodes 3 inositol monophosphatases involved in methionine
biosynthesis and halotolerance.
Correction for multiple alleles and allelic fragments left 130 haploid
ORFs potentially encoding Kex2p substrates. Of these, 14 are cited
genes whose protein products either are cleaved by Kex2p or contain
leader sequences with a putative Kex2p cleavage site:
SAP1-6, SAP8-9, XOG1, MP65,
HWP1, PHR1-2, and KEX2.
Each of the proteins encoded by these ORFs is homologous to a S. cerevisiae protein. Notably, two previously described SAPs were
not recovered by our script: Sap7p, which does not have a KR site, and
Sap10p, which does not have a leader sequence.
The 130 haploid ORFs were ascribed functions by a procedure involving
Blast of the candidate list against Genpept and HMMer of the list
against Pfam, followed by manual examination of the output (see
"Experimental Procedures"). 70 of the haploid ORFs have likely
S. cerevisiae homologues, and 60 do not. Of the 70 with
S. cerevisiae homologues, 58 (including the 14 cloned genes named above) have homologues with an ascribed name or function given by
the Saccharomyces Genome Database
(genome-www.stanford.edu/Saccharomyces/), including well characterized
extracellular S. cerevisiae proteins such as ScCrh1p
(Orf6.1231 of Assembly 6), ScSun4p (Orf6.2071), and ScExg2p
(Orf6.6664). The remaining 12 candidates are homologous to proteins of
unknown function in S. cerevisiae. The process and results
of classifying the ORFs is summarized in Fig.
4, and some of the proteins identified by
our script are presented in Table II. The
complete list of candidate Kex2p substrates can be accessed at the web
site, agabian.ucsf.edu.
C. albicans Has a Family of Acid Sphingomyelinases Not Found in S. cerevisiae--
Among the 60 haploid ORFs with no S. cerevisiae homologue, three fall into a particularly interesting
class of ORFs, which encode proteins not homologous to any S. cerevisiae protein, but with well-defined homologues in other
organisms. One of these (Orf6.7558) is homologous to a
Schizosaccharomyces pombe protein whose sequence has been
deposited in GenBankTM and ascribed a nucleoside permease
activity (NUP; gi 3764057). The remaining two proteins are homologues
of eukaryotic acid sphingomyelin phosphodiesterases (Orf6.3514 and
Orf6.7581) and have not been described in fungi. The two acid
sphingomyelinases identified as potential substrates of Kex2p are
members of a family of four C. albicans acid
sphingomyelinases (Fig. 5); the other two
possess no Kex2p cleavage site and are predicted to be intracellular. Some pathogenic bacteria secrete sphingomyelinases (see
"Discussion"), and the C. albicans sphingomyelinases may
similarly play a role in virulence and pathogenesis. With the exception
of the hypothetical enzyme in Ralstonia solanacearum,
bacterial sphingomyelinases are related to mammalian neutral
sphingomyelinases (34); these bear little sequence similarity to the
C. albicans enzymes.
One of the Candidate Cleavage Substrates of Kex2p Is Structurally
Related to Fungal Pheromones--
C. albicans is classified
as an imperfect yeast, although it possesses MAT C. albicans proteinase secretion, hyphal development,
egress from macrophages, and tissue invasion require kexin activity, allowing us to surmise that processing of proproteins by Kex2p is
essential for expression of the full range of virulence traits described for this pathogen. Inactivation of the kexin results in a
marked attenuation in C. albicans virulence; 1000 times more mutant yeast cells than wild-type cells are required to achieve an
equivalent ID50 at day 21 post-infection. Wild-type cells
form hyphae, which cause large pockets of tissue damage throughout the
kidney, while kex2/kex2 mutants are defective in
hyphal morphogenesis and tend to be restricted to the glomeruli.
Kidneys of mutant-infected mice suffer less tissue destruction; foci
are smaller and more discrete than those produced by wild type, and are
populated by fewer fungal cells. These results indicate that
kex2/kex2 cells are unable to effectively
negotiate the pathway from the bloodstream into and through renal
connective tissue. This defect might be attributable to more rapid
clearance from the circulation by phagocytic cells, a reduced capacity
to extravasate from the circulatory system, a decreased ability to
migrate through tissue spaces, or some combination thereof. These
possibilities are discussed below within the context of known and
potential C. albicans kexin substrates identified in the
present study.
The inability of C. albicans kex2/kex2 mutants to
exit the phagolysosomes of macrophages likely accounts for their
diminished virulence, as phagocytic clearance of the organism from the
circulation is the dominant host defense mechanism preventing systemic
candidiasis. Within minutes of intravenous injection, nonspecific
phagocytosis in the reticulo-endothelial system removes the majority of
C. albicans yeast from the bloodstream (40). A fraction of
ingested fungi may subsequently exit macrophages, probably through a
combination of enzymatic and mechanical means, placing them in a
position to invade deeper organs (25, 41). Two of the principal
virulence factors of C. albicans affected by inactivation of
Kex2p, hyphal formation and proteinase secretion (8), govern the
efficiency of exit from phagocytic cells (25, 26). Hyphal morphogenesis in C. albicans is regulated by different environmental cues
through at least four separate signal transduction pathways that
converge to induce the hyphal form (42).
kex2/kex2 cells are unable to form hyphae under
all conditions tested; therefore the morphogenic defect of the mutant
likely lies after the point where the pathways converge, perhaps
because the yeast fails to properly assemble one or more components
critical for the architecture of the hyphal wall. Consistent with this
hypothesis, we have observed irregularities in chitin deposition in
kex2/kex2 C. albicans (8). Kexin mutants of
another fungal pathogen, Candida glabrata, are
hypersensitive to Calcoflour, a dye that binds to chitin and is used to
identify cell wall mutants (43). S. cerevisiae
kex2 deletion mutants grown at low temperatures display
defects in cell polarity coincident with delocalization of actin and
chitin (16); these phenomena may reflect misprocessing of several cell
wall proteins, as judged by altered gel electrophoretic and isoelectric
focusing mobilities (44). A C. albicans exoglucansase likely
to be involved in cell wall remodeling, Xog1p, is probably processed by
Kex2p (45); however, xog1/xog1 mutants do not
have the grossly aberrant morphology of kex2/kex2
mutants (46). hwp1/hwp1 mutants, on the other
hand, do form stunted hyphae in renal tissue (47). Other cell wall proteins identified in the present study as being candidate Kex2p substrates, and thus possibly essential for hyphal formation, include
homologues of ScScw10p (Mp65), ScCrh1p, and ScSun4p; the last two are
potential glycosidases.
Unreported factors that may also contribute to escape from the
phagolysosome are the two putatively secreted sphingomyelinases identified in the present study. Sphingomyelinases of pathogenic bacteria, enzymatically similar but structurally unrelated to those of
C. albicans, lyse phagosomal membranes (48) or act as
hemolysins that facilitate the acquisition of iron from the host (49,
50). The C. albicans enzymes are structurally related to
mammalian secretory acid sphingomyelinases, which have been implicated
in inflammatory processes and possibly atherogenesis (51). In addition,
one of the products of sphingomyelinase catalysis, ceramide, is a
eukaryotic signaling molecule that controls key aspects of the
inflammatory reactions of macrophages, including apoptosis,
differentiation, and cytokine secretion (52, 53). Ceramide alters a
macrophage mitogen-activated protein kinase (MAPK) pathway, producing
an environment that is favorable for intracellular growth of
Leishmania donovani (54). Since phagocytosis by macrophages
of C. albicans, but not S. cerevisiae,
down-regulates macrophage MAPK activity (55), we hypothesize that
C. albicans alters host cell ceramide signaling pathways.
C. albicans in fact induces apoptosis in cells of monocyte
lineage (41, 56), an expected outcome of ceramide overproduction and
sphingomyelinase activity; however not all studies have confirmed this
(57).
The normal route of extravasation of C. albicans that escape
clearance by phagocytic cells is transcellular, involving
receptor-mediated attachment to endothelial cell surfaces coincident
with germ tube formation, entry into host cells via phagocytosis, and
exit facilitated by puncture of the host cell membrane by the hyphal
tip (58, 59). The informatic methods used in this study identified
several potential substrates of Kex2p that may participate in C. albicans adhesion to host cells. Several of these proteins are
rich in serine and threonine and, based on PSORT analyses, are
predicted to localize to the cell wall, properties common to yeast
agglutinins (60). Another group of kexin-processed substrates, the
SAPs, play a role in adhesion to epithelial cells (61). Cell wall mannoprotein Mp65, which experimentally is processed at a consensus kexin cleavage site, probably contributes to adhesion as it has a
complement-binding RGD site not found in its S. cerevisiae
homologue ScScw10p (62). The hyphal wall protein Hwp1p, on the other
hand, has been shown to be dispensable for attachment of the organism to endothelial cells (47).
kex2/kex2 mutants accumulate in kidney glomeruli
and the immediately surrounding tissue, indicating that cells that do
exit endothelial cells are compromised in their ability to invade solid tissue. The broad zones of tissue destruction and cellular displacement in the kidneys normally associated with wild-type C. albicans infections are absent in mice infected by the mutant, and
are replaced by compact lesions. This likely reflects the inability of
the mutants to form hyphae, and is consistent with the traditional view
that the hypha is a principal effector of pathogenesis (63). The
migration pathway of wild-type C. albicans into deeper
tissue presents a leading edge of hyphae that mechanically dislodge
desmosomes and enzymatically degrade ECM (64);
kex2/kex2 mutants engage in none of these
behaviors. The invasiveness of the mutant, while greatly reduced, is
not entirely abolished, as a few yeast do enter the kidney parenchyma.
These exceptions are consistent with the observation that translocation
of the yeast form can be a minor component of C. albicans
spread (65), and that invasion is to some degree effected by mechanisms
independent of hyphae, such as the release of several hydrolases (66,
67).
In sum, the present study demonstrates that, while not essential for
viability in vitro, kexin activity is required for full virulence of C. albicans. At minimum, the enzyme is involved
in a variety of functions that manifest at the host-pathogen interface: proteinase secretion, hyphal development, escape from macrophages, and
invasion of solid tissue. The invasion defect of
kex2/kex2 mutants reminds us of tumor cells whose
metastasis is attenuated by a specific inhibitor of furin, a homologue
of Kex2p found in metazoans (2), and reflects the role of this enzyme
in mediating the maturation of proteins that interact with the
environment. Our informatic method identified several secreted
hydrolases, cell wall constituents, adhesins, and other proteins that
are known to make important contributions to C. albicans
pathogenesis, and discovered a larger set whose contribution remains to
be assessed. In aggregate this list identifies a subset of potential
kexin substrates, which ultimately function at the host-pathogen
interface. Since homologues of Kex2p are likely to play significant
roles in the biology of eukaryotic pathogens in general, and in light of the increasing number of genomes being sequenced, it should prove
useful to apply this computational approach to search for similar
proteins in other pathogens. Demonstrations that the polyprotein allergens of parasitic nematodes and the major adhesin of the pathogenic fungus Coccidioides immitis are processed by
kexins (68, 69) support this conjecture. Some of the substrates of unknown function, which we identified by this procedure may mediate previously unsuspected interactions between parasitic organisms and
their hosts, between individuals of a colonizing population, and
between different species or populations of microbes.
-factor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-type mating
prepropheromones (7), killer toxins (7), zymogens of
secreted proteinases (8-10), lipases (11), polysaccharide-degrading
enzymes (12, 13), and themselves (14). Deleting the kexin of the yeast
Yarrowia lipolytica abolishes the formation of hyphae (15).
kex2-null mutants of the yeast Saccharomyces
cerevisiae are viable but exhibit conditional morphological
abnormalities (16), defective vacuolar proton-translocating V-ATPase
activity (17), cold-sensitive growth (16), and a partial defect in
meiosis (genome-www.stanford.edu/Saccharomyces). Genetic data indicate
that Kex2p activity somehow may influence the RNA polymerase II complex
(18).
-factor and other fungal pheromones that require processing by kexin for activation. The identification of a potential substrate of Kex2p, which is structurally similar to
-factor, is
consistent with recent studies that show that
cell derivatives of
the kex2/kex2 strain are unable to mate with
a cells, while kex2/kex2 a
cells mate at wild-type efficiency with
cells.2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ura3::imm434/
ura3::imm434 KEX2/kex2::hisG-URA3-hisG, and 3) CNA3,
ura3::imm434/
ura3::imm434 kex2::hisG/kex2::hisG-URA3-hisG.
Strains CNA1 and CNA3 were derived from CAI4, a
ura3/ura3 derivative of SC5314. Strains CNA3-1
and CNA3-2 are isogenic, originating from different transformations (8). Yeast forms were routinely maintained on YEPD plates (2% bactopeptone, 1% yeast extract, 2% glucose, 2% agar [DIFCO]) at 30 °C. Hyphae were induced in serum or Lee's media following
standard procedures. To embed cells in agar, strains were grown for
48 h in YEPD at 30 °C and washed twice with distilled water;
~100 colony-forming units (CFU) were mixed with 20 ml of YPS-agar
(1% yeast extract, 2% bactopeptone, 2% sucrose, 1% agar), plated in 90-mm petri dishes (19), and incubated at 25 °C. To enhance visualization of colony morphology, plates were stained with 0.1% Amido Black in acetic acid/methanol/water (10:25:65) and destained with
acetic acid/methanol/water (10:20:70).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ID50 of DBA/2N mice inoculated with wild-type,
KEX2/kex2, and kex2/kex2 strains of C. albican
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Fig. 1.
Foci of SC5314 (wild-type) and CNA3
(kex2/kex2) C. albicans
in the kidneys of infected mice 2-day post-infection.
Hematoxylin and eosin-stained sections were further enhanced
with periodic acid-Schiff to highlight the morphology of the fungi.
A, micrograph depicting the broad lesions caused by SC5314,
where host tissue is visibly disrupted and the infecting fungi
proliferate as hyphae; B, contrasting view of foci of CNA3
cells, where cells are often found in the glomeruli (G) and
colonies contain fewer cells; C, similar view of uncolonized
kidney and glomeruli. Higher magnification of fungal lesions,
highlighting the difference of morphology between SC5314 (D)
and CNA3 (E). Arrows indicate fungal cells.
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Fig. 2.
C. albicans interactions with cultured
P388D1 cells. A, SC5314 4-h post-infection. B,
CNA3 4-h post-infection. C, CNA3 24-h post-infection.
Macrophage cultures co-incubated for 24 h with SC5314 were almost
entirely cleared, with C. albicans growing as masses of
hyphae. Arrows indicate fungal cells.
phenotype.
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Fig. 3.
Micrographs of colonies growing in soft agar.
A, SC5314 grown for 6 days at 25 °C embedded in YPS agar;
B, CNA3 grown for 3 days at 25 °C embedded in YPS agar;
C, typical morphology of edges of SC5314 colony embedded in
agar; D, typical edge of CNA3 colony viewed at the same
magnification as (C). E, same as (A)
with rare filamentous sector indicated by arrow.
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Fig. 4.
Flowchart describing the extraction of
potential Kex2p cleavage substrates from the C. albicans
genome, and their classification. See text for details of
computational and informatic procedures.
Selected C. albicans proteins computationally identified as potential
cleavage substrates of Kex2p
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Fig. 5.
Dendrogram of the C. albicans
family of acid sphingomyelinases (aSMases).
The species labels are as follows: Bc (Bacillus
cereus), Rs (Ralstonia solanacearum),
Hs (Homo sapiens), Ca (Candida
albicans). The C. albicans proteins identified as
possible Kex2p cleavage substrates are highlighted in bold
face. The sequences were aligned, and the N-J tree was calculated
using ClustalX 1.8 (www-igbmc.u-strasbg.fr/BioInfo/ClustalX/).
- and
MATa-like mating-type loci (35). The genome also encodes
many gene products consistent with a complete sexual cycle (36).
C. albicans strains engineered to be homozygous for the
mating-type locus are able to mate with strains similarly engineered at
the a mating-type locus (35, 37). Deletion of
KEX2 abrogates the ability of
cells to mate with
a cells, but not the ability of a cells to mate
with
cells,2 suggesting that C. albicans
cells may secrete an
-specific mating pheromone processed by Kex2p.
Although well-characterized peptide mating pheromones such as the
-factor of S. cerevisiae and the P-factor of S. pombe are cleaved by kexins (7, 38), our semiautomated annotation
of the list of candidate Kex2p cleavage substrates did not describe a
protein having a significant degree of sequence similarity to known
fungal pheromones. This is not surprising, as fungal pheromones such as
-factor and P-factor do not bear much sequence similarity to one
another (see Fig. 6). To ask whether
C. albicans synthesizes peptide-mating pheromones, we
elaborated our script to query the C. albicans genome for
proteins that possess structural commonalities with
-factor and
P-factor in addition to a leader sequence and a Kex2p cleavage motif.
The algorithm computationally cleaved the candidate proteins at the Kex2p cleavage sites and compared the lengths of the resulting peptide
fragments. Eighteen candidates were cleaved into at least one pair of
equally sized fragments. Of these, a single protein (Orf6.4306) also
possesses the X(A/P)X(A/P) motif characteristic of
-factor and P-factor (Fig. 6). This dipeptide motif is recognized and processed by the S. cerevisiae ScSte13p aminopeptidase,
and a clear homologue of ScSte13p is present in the C. albicans genome (Orf6.4953). Also found in Assembly 6 are C. albicans homologues to other S. cerevisiae proteins
related to
-factor synthesis and signaling, including ScKex1p
(peptidase involved in
-factor maturation; Orf6.7946) and ScSte2p
(receptor for
-factor; Orf6.4012) (36). The transcript of Orf6.4306
is specifically expressed in the opaque switch phenotype of the WO-1
strain of C. albicans.3 Mating
competent cells in strain SC5314 have an opaque-like phenotype, and
demonstrate morphological behavior reminiscent of the
pheromone-dependent "schmoos" of S. cerevisiae
(39).
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Fig. 6.
Sequence comparison of the putative C. albicans prepropheromone (Orf6.4306) to those of the related
ascomycota S. cerevisiae and S. pombe. The mature pheromones are distinguished and
aligned by spacing, the Kex2p cleavage recognition sites by
underlining. The Kex2p sites and mature peptides have been
confirmed experimentally in S. cerevisiae and S. pombe but not in C. albicans.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Jeff Roberts and David Blake for technical assistance, and Stew Scherer for advice. We also thank our collaborators Ted Jones, Nancy Federspiel, Curtis Palm, Donna Bowe, and Ron Davis at the Stanford Genome Technology Center for the generosity in providing the C. albicans genome sequence prior to its publication.
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FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grants A133317, RO1DE12940, P01 DE07946-15S2, and PO1 DE07946.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ Supported by University of California University-wide AIDS Research Program Grant F97-SF-048.
** Present address: Incyte Genomics, Palo Alto, CA 94304.
To whom correspondence should be addressed: Dept. of
Stomatology, University of California, 521 Parnassus Avenue, Box 0422, San Francisco, CA 94243-0422. Tel.: 415-476-6845; Fax: 415-476-0664; Email: agabian@itsa.ucsf.edu.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209713200
2 Magee, B. B., Legrand, M., Alarco, A.-M., Raymond, M., and Magee, P. T. (2002) Mol. Microbiol. 46, 1345-1351.
3 Lan, C.-Y., Newport, G., Murillo, L. A., Jones, T., Scherer, S., Davis, R. W., and Agabian, N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 14907-14912.
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; ORF, open reading frame; CFU, colony-forming unit; MAPK, mitogen-activated protein kinase.
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REFERENCES |
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