Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, University of Aberdeen, Aberdeen AB25 2ZD, UK1
Department of Bacteriology and Mycology, Janssen Research Foundation,B-2340 Beerse, Belgium2
Author for correspondence: Frank C. Odds. Tel: +44 1224 273128. Fax: +44 1224 273144. e-mail: f.odds{at}abdn.ac.uk
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ABSTRACT |
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Keywords: Candida albicans, experimental infection, virulence
Abbreviations: MI, morphology index
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INTRODUCTION |
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We were interested in devising an approach to experimental intravenous C. albicans infection that facilitated differentiation of pathological effects dependent on host factors from effects related to the infecting strain. We therefore experimented with three C. albicans isolates, including one known to be highly attenuated in virulence which formed germ tubes poorly in serum. The fungi were injected intravenously into two animal types, female mice and male guinea pigs, deliberately chosen to maximize any host-related differences in progression of the infection. Preliminary experiments have shown that the pattern of infection seen after injection of C. albicans intravenously in guinea pigs differed markedly from the pattern in similarly infected outbred mice (Agabian et al., 1994 ; Buurman et al., 1998
). In particular, the organ distributions of C. albicans differed with time between the two hosts, and fungal cells recovered from mouse tissues by KOH treatment formed extensive true hyphae, whereas hyphae were seldom seen in guinea pig tissues.
Since growth conditions for C. albicans can influence properties such as surface hydrophobicity of a challenge inoculum and hence lethality for animals (Antley & Hazen, 1988 ), we first investigated whether inocula grown at the same temperature but in two peptone-based broth media of considerably different composition also showed any medium-related difference in lethality. We designed these dose versus medium versus survival time experiments to reveal the maximum extent of quantitative inter-experiment variability in challengesurvival tests. In a second series of experiments, animals were infected with inocula that gave the same mean survival time regardless of inherent lethality differences in the test strains. Under these conditions, the morphological forms of the fungus in deep organs varied between C. albicans strains as well as between animal hosts, but the gross fungus burdens recovered from deep organs were mainly host-dependent. The use of survival-standardized inocula provides a means for future experiments to distinguish experimentally between fungus factors that are important for virulence at the time of intravenous challenge and those that relate to later pathogenic events.
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METHODS |
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All three yeast strains were maintained on Sabouraud glucose agar (Oxoid). For the preparation of inocula, a yeast colony was gently sampled with a wet inoculating loop and yeasts were transferred to 5 ml inoculation medium in a sterile, 15 mm glass test tube. Two inoculation media were used: one was Sabouraud broth (mycological peptone, Oxoid, 10 g l-1; glucose, 40 g l-1) and the other was CYGi, a 10-fold dilution of another peptone-glucose medium previously described (Odds, 1991 ) and comprising pancreatic casein digest (Merck), 3·0 g l-1; yeast extract (Difco Laboratories), 3·0 g l-1; glucose, 6·0 g l-1; MOPS (Acros Organics), 10 g l-1; and Tris (Acros), 3·0 g l-1; pH 7·2, sterilized by autoclaving.
The test tube cultures were incubated in wheels rotating at 20 r.p.m. set at an angle of 5° to the horizontal in an incubator at 30±1 °C (Odds, 1991 ). For measurement of growth rates, the turbidity of the cultures was determined spectrophotometrically at 570 nm at intervals up to 24 h after inoculation. For preparation of inocula, the cultures were incubated for 1820 h at 30 °C, then the cell concentration was estimated by means of haemocytometer counts. The morphology of all three strains in both media was spheroidal yeast cells without pseudohyphal or hyphal forms. The yeast suspensions were diluted in sterile physiological saline as required. The suspensions contained >95% viable cells as judged by phase-contrast microscopy. A sample of each suspension used for infection was serially diluted and plated on Sabouraud agar to determine the numbers of c.f.u. ml-1. For comparisons between host species, infecting inocula were expressed as c.f.u. (g body weight)-1.
For examination of C. albicans morphology in vitro, yeasts were grown overnight in CYGi broth at 30 °C with constant rotation and cell suspensions were diluted to 15x105 yeast cells ml-1 in pre-warmed volumes of foetal calf serum, buffered Eagles modified essential medium (EMEM; Odds et al., 1985 ) or modified Sabouraud broth (MSAB; Evans et al., 1975
). These cultures were incubated at 37 °C (serum and EMEM) or 40 °C (MSAB). Samples were removed for microscopic observation and scored for morphological form after 2, 4 and 6 h incubation.
Experimental infections.
Specific pathogen-free male albino guinea pigs and female NMRI mice were the animal hosts. Pirbright albino guinea pigs (Charles River Associates, Kisslegg, Germany) were housed individually and mice (Charles River Associates, Brussels, Belgium) in groups of up to ten. They were maintained under conditions approved by the Animal Welfare Committee of the Janssen Research Foundation and were fed food and water ad libitum. Individual animals were weighed and infected intravenously with C. albicans via either the lateral tail vein (mice) or the lateral vein of the penis (guinea pig). In a first series of experiments, animals in groups of five were infected with different concentrations of the three strains of C. albicans, grown in Sabouraud or CYGi broth, to establish the relation between infecting dose and mean survival time. To investigate the extent of quantitative variations in survival time, a schedule was devised that involved randomization of dosages, strains and animal hosts so that on each day when an experiment was set up, a subset of combinations of strain, medium, inoculum size and animal host was chosen randomly. In this way, no more than one concentration of any individual C. albicans strain from Sabouraud or CYGi medium was inoculated in the same host in the same experimental run. The day of death of each infected animal (or day when animals in moribund condition were killed) was recorded and observations were continued up to 21 d after challenge. The experiments were all set up within a 3-week period and new batches of experimental animals of approximately the same age were purchased each week.
In a second series of experiments, guinea pigs and mice were again infected with the three strains of C. albicans, grown in the two different media, but inoculum sizes were judged from the results of the first experiments to give a predicted survival time of 14 d in all cases. This time was chosen to allow for infection to progress in deep organs without distortions of the infection pattern resulting from acute deaths. On days 1, 2, 3, 4, 7 and 14 after challenge, three animals from each experimental group were killed. The left and right kidneys, liver, lungs, spleen and brain were removed under aseptic conditions and a portion of each organ was weighed and placed in 6 ml sterile physiological saline. These samples were homogenized in an ultra-Turrax-type apparatus (Janke and Kunkel) and serial dilutions were plated on Sabouraud agar for determination of C. albicans c.f.u. (g organ)-1. A further 2 ml of each homogenate was mixed with 2 ml 60% (w/v) KOH solution to permit recovery of C. albicans cells for morphological examination. The mixtures were incubated for 2 h at 30 °C with gyratory shaking at 100 r.p.m., centrifuged at 1500 g for 10 min, and the supernatant fluid was removed. The pellet was washed in distilled water three times by repeated resuspension and centrifugation and finally resuspended to a volume of approximately 0·5 ml in water for microscopic examination of the morphology of the recovered C. albicans cells.
Estimation of C. albicans morphological forms in vitro and in vivo.
To estimate the nature and distribution of C. albicans cell shapes recovered from infected tissues or grown in vitro, an adaptation of the morphology index, MI (Merson-Davies & Odds, 1989 ) was used. Cell units with a spherical or nearly spherical shape were scored 1; cell units with an ovoid shape and a length up to twice the diameter of the cell were scored 2; cell units with a pseudohyphal appearance (obvious constrictions at septal junctions) and with a length more than twice the cell diameter were scored 3; and parallel-sided hyphal cell units with minimal constrictions at septal junctions were scored 4. For each microscopic field examined, the numbers of cells with shape score 1, 2, 3 or 4 were recorded and the process was repeated, when sufficient cells were available for scrutiny, until at least 50 cell units had been examined and scored. When this system was used for C. albicans grown in vitro, only newly emergent growth forms were assessed; the morphology of parent blastoconidia was not scored; 100 cell units were scored for cultures in vitro.
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RESULTS |
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All three strains formed germ tubes in foetal calf serum at 37 °C. By 4 and 6 h, 100% of B2630 and SC5314 cells appeared as characteristic parallel-sided hyphal outgrowths, mean MI=4·0. RV4688, by contrast, formed true hyphae less readily in serum. Mean MI±SD for this strain was 2·2±1·0 at 2 h, 2·8±1·2 at 4 h and 2·7±1·0 at 6 h (n=100 for each estimation). The proportion of cell outgrowths with MI=4 was 9% at 2 h, 43% at 4 h and 31% at 6 h. RV4688 was therefore a strain with a highly mixed morphological phenotype in serum. In EMEM and MSAB, only B2630 and SC5314 formed true hyphae, with MI=4 at 4 h for >95% of new outgrowths, respectively. For RV4688, after 4 h incubation in EMEM or MSAB, there were no true hyphal outgrowths formed: approximately 50% of outgrowths were buds with MI=1, 40% long buds with MI=3 and 10% pseudohyphal forms with MI=3.
Mean survival times of mice and guinea pigs with disseminated C. albicans infection
Fig. 1 shows the results of experiments in which yeasts of the three strains of C. albicans, grown in either Sabouraud or CYGi broth, were injected intravenously at different concentrations. Each mean±SEM data point in each dose-response curve was the result of a separate experiment. The results indicated considerable quantitative variation between runs (individual data points) and between animals (SEMs for each point), despite which the lethality trends for each strain tested were clear and unequivocal. In both mice and guinea pigs, SC5314 and B2630 showed similar lethality and RV4688 was markedly attenuated in virulence for both animal hosts. The dosesurvival time curves for all three strains depended on the culture medium used to grow the yeasts that were injected, with inocula from Sabouraud broth cultures consistently leading to shorter survival times, dose for dose, than inocula from CYGi broth cultures.
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Tissue distributions of C. albicans and fungal morphology in tissues of animal hosts with disseminated infection
Fig. 2 shows the logarithms of counts of viable C. albicans determined for mice and guinea pigs infected with C. albicans challenge doses chosen to result in mean survival times of 14 d. The results shown are from experiments where the yeast inoculum was grown in Sabouraud broth. Essentially the same results were obtained when the inoculum was grown in CYGi: when challenge inocula were adjusted to achieve the same levels of infectivity, no differential effects of culture medium were apparent on the distribution of cells in major organs.
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In guinea pigs (Fig. 2df), the progression of tissue burdens after intravenous challenge with all three C. albicans strains differed from those in mice. In this host, c.f.u. recovered from brain tissue were uniformly equally high as those recovered from the right and left kidneys. The highest tissue burdens were determined for RV4688 (Fig. 2e)
, but the general pattern of changes in tissue burdens with time were essentially similar for all three strains regardless of the growth medium used to prepare the challenge dose. Highest counts of C. albicans were recovered from brain and kidney samples on day 4 or day 7 after challenge, with a marked decrease in the c.f.u. g-1 in brain and kidney samples by 14 d after challenge. Counts of viable fungi from liver, spleen and lung samples all followed a similar trend, with a tendency towards decreasing c.f.u. g-1 from day 3 post-challenge onwards, with the lowest counts recovered in animals still alive at the predicted mean survival time of 14 d.
Morphology of C. albicans recovered from infected tissues
In tissue samples where fewer than 103 c.f.u. g-1 were recovered in culture, too few C. albicans cells were seen in the pellets of fungi recovered after KOH treatment to permit estimation of morphology scores on a sample size of at least 50 cells. Only for samples from kidneys (both animal hosts) and brain tissues (guinea pigs) was there a consistently high number of fungal cells available for morphology scoring to permit analysis of fungal morphology at different times after challenge. The data in Fig. 3 show that C. albicans true hypha formation in the tissues (percentage of cells with MI 4) varied both with the infecting fungus strain and the animal host but was not dependent on the growth medium used to prepare the inoculum.
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B2630. In samples of guinea pig kidney tissue, mixed fungal morphologies were found at all times after i.v. challenge, and hyphal forms became rare from 3 d after challenge (Fig. 3a). Elongated yeast cells (MI=2) and pseudohyphal forms (MI=3) predominated in samples from day 4 onwards, which is why the proportion of cells with MI 4 in Fig. 3
falls below 50%. In guinea pig brain samples, the distribution of morphologies showed a preponderance of hyphal forms on days 13 (Fig. 3b)
, with a decreasing proportion of such forms later in the course of infection. Among mice infected with B2630, kidney samples showed increasing hyphal predominance among fungal morphologies recovered over the course of infection, similar to the results obtained with SC5314 (Fig. 3c)
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RV4688. The predominant morphological form in guinea pig kidneys and brain and in mouse kidneys from animals infected with this C. albicans strain was the spheroidal yeast cell. In kidney and brain samples from guinea pigs, true hyphal forms (MI=4) were recorded for only four fungus cells out of more than 4000 sampled (Fig. 3). However, in samples from kidneys of mice infected with RV4688, significant proportions of true hyphal forms were seen on days 7 and 14 after challenge (Fig. 3c)
, even though cells with morphology score 1 remained predominant.
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DISCUSSION |
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Our results in animal lethality tests (Fig. 1) show that, even though gross differences in virulence between strains can be demonstrated by challenge experiments with inocula prepared in a single medium, the quantitative variation in survival rates is considerable both between animals and between experiments. This suggests that the reproducibility of lethality differences measured between parent and mutant strains of C. albicans may be more reliably established from experiments with randomized challenge dose designs, similar to that used in the present study, rather than in single experiments, especially when those differences are not substantial.
This is the first study in which differences in lethality have been broken down into (a) effects related to the preparation of the inoculum, (b) the challenge dose normalized for inherent differences in the virulence of individual strains and (c) two different host species. This approach facilitated the distinction between progression of infection as related to individual host tissues and infective effects related specifically to the infecting strain. While the growth medium influenced mean survival times in simple dosesurvival titration experiments, when the challenge inocula were normalized to equalize survival times, no influence of growth medium was evident in terms of organ burdens of C. albicans or in fungal morphologies in infected tissues at various times after challenge. These findings suggest that although the growth conditions for a C. albicans infecting inoculum are an important factor in determining a strains ability to gain an initial invasive hold immediately after injection into an animal host, they are unimportant in the subsequent development of infection.
The quantitative C. albicans organ burdens measured at times after challenge were similar for all three strains of the fungus, but differed between the two animal hosts tested (Fig. 2). This suggests that host factors play a more important role in determining quantitative tissue burdens than any virulence factors in the fungus, once infection has been established in tissues by administration of a suitably large inoculum. Several prior studies have shown that, in a murine host, the genetic background of the mouse strain determines not just overall susceptibility to intravenous C. albicans challenge, but also the susceptibility of individual host tissues to infection by circulating fungus cells (Ashman & Bolitho, 1993
; Ashman et al., 1991
, 1996
, 1997
; Fulurija et al., 1996
). The mouse gene products that regulate the susceptibility of individual tissues to the fungus have been designated Carg1p and Carg2p (Ashman et al., 1997
). Gender is a further significant influence on susceptibilities of mice of C. albicans challenge (Ashman et al., 1991
). In our experiments, the hosts used were of different genders, as well as different species, a deliberate choice to emphasize the considerable differences effected by host factors in progression of experimental disseminated C. albicans infections. Ashman et al. (1997
) observed extensive fungal colonization of brain tissue and severe brain lesions only in four of 15 recombinant inbred mouse strains derived from two progenitor strains. Neutrophil responses have been shown to influence differences in susceptibility of mouse brains and kidneys to C. albicans infection (Fulurija et al., 1996
). Our finding that brain burdens of C. albicans matched those of kidneys in guinea pigs but not in mice is consistent with these previous reports of differences in animal host susceptibility to cerebral infection with the fungus.
While our data show a strong animal host-dependency of the fungal burdens in specific tissues, the morphological forms of the fungi recovered from the tissues by KOH treatment were clearly a function of the characteristics of the infecting strain. Two of our three C. albicans strains formed true hyphae readily under different, well-characterized environments known to favour filamentous growth in vitro, whereas RV4688 formed true hyphae only rarely and reluctantly in vitro. In the kidneys of both animal species and the brains of guinea pigs, each of the three C. albicans strains appeared to follow an individual course of morphological development, but with characteristics that also indicated at least a partial influence from the host tissue micro-environment (Fig. 3). In mouse kidneys, the morphological trend for all three strains was for the proportion of true hyphal cells to increase with time. In guinea pig kidneys, by contrast, proportions of hyphal cells were high soon after challenge (except for RV4688) and they fell over the course of infection. These trends therefore appeared to be attributable to the host tissues. RV4688 hardly ever formed hyphae in vivo, behaviour consistent with its hypha formation in vitro: it started to form measurable proportions of true hyphae only in mouse kidney tissues by days 7 and 14 after challenge.
In general, true mycelial forms proliferated in mouse kidneys while elongated yeasts and pseudohyphal forms predominated in guinea pig kidneys. Morphological differences of this nature were noted en passant but not specifically reported by Winblad (1975) in his comparative histopathological study of C. albicans infection in the kidneys of mice and guinea pigs. The figure legends in Winblads paper describe the fungi as mainly in the mycelial phase in mouse kidney sections and as both yeast and mycelial phase in guinea pig kidney sections. The histopathological forms illustrated by Fransen et al. (1984
) in guinea pig tissues similarly suggest a predominance of yeast forms and pseudohyphae, rather than true hyphae, in most C. albicans-infected tissues. De Bernardis et al. (1993)
described a C. albicans strain, CA-2, which formed pseudohyphae in vivo in a rat vaginal model of infection, but was unable to form germ tubes in vitro. Those observations, plus those of the present study, indicate that morphogenesis of C. albicans is a highly complex response of the fungal cells to their micro-environment, with no obligate correlation between a strains ability to form filaments in vivo and in vitro.
These observations on morphological development of the three strains in vivo generally support the traditional view of C. albicans which implicates formation of hyphae as a property conducive to pathogenicity (Kobayashi & Cutler, 1998 ; Lo et al., 1997
; Odds, 1988
; Brown & Gow, 1999
) since the strain that poorly formed hyphae was the least virulent in terms of gross lethality per challenge dose. However, in the present experiments when the strain reluctant to form hyphae was injected at a concentration aimed to give the same mean survival time as in the animals infected with the prolific hypha-formers, its tissue burdens after infection were as high as or even higher than those of the other two strains (Fig. 2)
and they followed the same course of changes with time. The pathological factors that ultimately led to death of animals infected with C. albicans are unknown, but they were clearly not absolutely dependent on the capacity of the infecting strain to form hyphae: however, the attenuated lethality of RV4688, dose for dose, may indicate that an ability to form hyphae enhances the initial ability of a strain to achieve a pathologically significant hold on host tissues at or soon after the time of intravenous challenge.
Older studies have shown clearance of almost all C. albicans cells from the blood of experimental animal hosts within only minutes of intravenous injection (Baine et al., 1974 ; Iannini et al., 1977
; Jeunet et al., 1970
; Rink et al., 1981
; Sawyer et al., 1976
). These reports indicate that immediate, nonspecific phagocytosis of the fungus cells by the reticulo-endothelial system is a potent and for the fungus critical encounter between host and pathogen in models that involve direct intravenous challenge. If overwhelming numbers of a weakly virulent strain are injected to facilitate survival of yeast cells that are not inherently well-equipped to resist immediate host clearance mechanisms, as in our experiments with RV4688, then the surviving yeasts are evidently capable of expressing sufficient virulence to set up pathologically significant tissue burdens, a process that our data show is independent of hypha formation.
The number of studies investigating the influence of specific gene deletions on virulence in C. albicans has grown extensively in recent years: most of these publications base their conclusions on the gross lethal outcome of intravenous challenge experiments in mice. The present study adds weight to existing experimental evidence that shows that the intrinsic virulence of C. albicans is modulated by both the strain of animal host chosen for lethality testing and the conditions used to generate yeast challenge inocula. Despite the existence of such prior evidence, the lethality testing of gene knockouts in C. albicans has been done in a diverse range of mouse strains and the medium, temperature and time used to prepare each inoculum differ from study to study: these variables have not even been detailed in several such publications.
Our study has confirmed and extended the evidence that C. albicans intravenous virulence in vivo is dependent on the animal species challenged and the growth conditions used for the challenge inoculum, as well as on differences between fungal strains. Our use of survival-standardized inocula offers a novel approach for future investigations to differentiate early- and late-stage host and pathogen factors involved in the infectious disease process with this fungus. We hope that future experiments on the pathogenesis of experimental C. albicans infections will take greater account of the sources of variability we have highlighted in our work, as well as extending to include models that take account of the mucosal barriers to dissemination of infection usually found in the human setting.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Antley, P. P. & Hazen, K. C. (1988). Role of yeast cell growth temperature on Candida albicans virulence in mice. Infect Immun 56, 2884-2890.[Medline]
Ashman, R. B. & Bolitho, E. M. (1993). Strain differences in the severity of lesions in murine systemic candidiasis correlate with the production of functional gamma interferon by Candida-activated lymphocytes in vitro. Lymphokine Cytokine Res 12, 471-476.[Medline]
Ashman, R. B., Kay, P. H., Lynch, D. M. & Papadimitriou, J. M. (1991). Murine candidiasis: sex differences in the severity of tissue lesions are not associated with levels of serum C3 and C5. Immunol Cell Biol 69, 7-10.[Medline]
Ashman, R. B., Fulurija, A. & Papadimitriou, J. M. (1996). Strain-dependent differences in host response to Candida albicans infection in mice are related to organ susceptibility and infectious load. Infect Immun 64, 1866-1869.[Abstract]
Ashman, R. B., Fulurija, A. & Papadimitriou, J. M. (1997). Evidence that two independent host genes influence the severity of tissue damage and susceptibility to acute pyelonephritis in murine systemic candidiasis. Microb Pathog 22, 187-192.[Medline]
Baine, W. B., Koenig, M. G. & Goodman, J. S. (1974). Clearance of Candida albicans from the bloodstream of rabbits. Infect Immun 10, 1420-1425.[Medline]
Becker, J. M., Henry, L. K., Jiang, W. & Koltin, Y. (1995). Reduced virulence of Candida albicans mutants affected in multidrug resistance. Infect Immun 63, 4515-4518.[Abstract]
Brown, A. P. J. & Gow, N. A. R. (1999). Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol 7, 333-338.[Medline]
Bulawa, C. E., Miller, D. W., Henry, L. K. & Becker, J. M. (1995). Attenuated virulence of chitin-deficient mutants of Candida albicans. Proc Natl Acad Sci U S A 92, 10570-10574.[Abstract]
Buurman, E. T., Westwater, C., Hube, B., Brown, A. J., Odds, F. C. & Gow, N. A. R. (1998). Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans. Proc Natl Acad Sci U S A 95, 7670-7675.
Calera, J. A., Zhao, X. J., De Bernardis, F., Sheridan, M. & Calderone, R. (1999). Avirulence of Candida albicans CaHK1 mutants in a murine model of hematogenously disseminated candidiasis. Infect Immun 67, 4280-4284.
Calera, J. A., Zhao, X. J. & Calderone, R. (2000). Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans. Infect Immun 68, 518-525.
Csank, C., Schroppel, K., Leberer, E., Harcus, D., Mohamed, O., Meloche, S., Thomas, D. Y. & Whiteway, M. (1998). Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis. Infect Immun 66, 2713-2721.
De Bernardis, F., Adriani, D., Lorenzini, R., Pontieri, E., Carruba, G. & Cassone, A. (1993). Filamentous growth and elevated vaginopathic potential of a nongerminative variant of Candida albicans expressing low virulence in systemic infection. Infect Immun 61, 1500-1508.[Abstract]
De Bernardis, F., Muhlschlegel, F. A., Cassone, A. & Fonzi, W. A. (1998). The pH of the host niche controls gene expression in and virulence of Candida. Infect Immun 66, 3317-3325.
Diez-Orejas, R., Molero, G., Navarro-Garcia, F., Pla, J., Nombela, C. & Sanchez-Perez, M. (1997). Reduced virulence of Candida albicans MKC1 mutants: a role for mitogen-activated protein kinase in pathogenesis. Infect Immun 65, 833-837.[Abstract]
Evans, E. G. V., Odds, F. C. & Holland, K. T. (1975). Resistance of the Candida albicans filamentous cycle to environmental change. Sabouraudia 13, 231-238.[Medline]
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717-728.
Fransen, J., Van Cutsem, J., Vandesteene, R. & Janssen, P. A. J. (1984). Histopathology of experimental systemic candidosis in guinea-pigs. Sabouraudia 22, 455-469.[Medline]
Fulurija, A., Ashman, R. B. & Papadimitriou, J. M. (1996). Neutrophil depletion increases susceptibility to systemic and vaginal candidiasis in mice, and reveals differences between brain and kidney in mechanisms of host resistance. Microbiology 142, 3487-3496.[Abstract]
Gale, C. A., Bendel, C. M., McClellan, M., Hauser, M., Becker, J. M., Berman, J. & Hostetter, M. K. (1998). Linkage of adhesion, filamentous growth, and virulence in Candida albicans to a single gene, INT1. Science 279, 1355-1358.
Ghannoum, M. A. (1998). Extracellular phospholipases as universal virulence factor in pathogenic fungi. Nippon Ishinkin Gakkai Zasshi 39, 55-59.[Medline]
Ghannoum, M. A., Spellberg, B., Saporito-Irwin, S. M. & Fonzi, W. A. (1995). Reduced virulence of Candida albicans PHR1 mutants. Infect Immun 63, 4528-4530.[Abstract]
Hube, B., Sanglard, D., Odds, F. C., Hess, D., Monod, M., Schafer, W., Brown, A. J. & Gow, N. A. R. (1997). Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence. Infect Immun 65, 3529-3538.[Abstract]
Iannini, P. B., Arai, G. D. & Laforce, F. M. (1977). Vascular clearance of blastospore and pseudomycelial phase Candida albicans. Sabouraudia 15, 201-205.[Medline]
Jeunet, F. S., Meuwissen, H. J. & Good, R. A. (1970). Fate of Candida albicans in neonatally thymectomized rats. Proc Soc Exp Biol Med 133, 53-56.
Jiang, W., Gerhold, D., Kmiec, E. B., Hauser, M., Becker, J. M. & Koltin, Y. (1997). The topoisomerase I gene from Candida albicans. Microbiology 143, 377-386.[Abstract]
Kobayashi, S. D. & Cutler, J. E. (1998). Candida albicans hyphal formation and virulence: is there a clearly defined role? Trends Microbiol 6, 92-94.[Medline]
Kvaal, C. A., Srikantha, T. & Soll, D. R. (1997). Misexpression of the white-phase-specific gene WH11 in the opaque phase of Candida albicans affects switching and virulence. Infect Immun 65, 4468-4475.[Abstract]
Lay, J., Henry, L. K., Clifford, J., Koltin, Y., Bulawa, C. E. & Becker, J. M. (1998). Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies. Infect Immun 66, 5301-5306.
Leberer, E., Ziegelbauer, K., Schmidt, A., Harcus, D., Dignard, D., Ash, J., Johnson, L. & Thomas, D. Y. (1997). Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p. Curr Biol 7, 539-546.[Medline]
Leidich, S. D., Ibrahim, A. S., Fu, Y. & 8 other authors (1999). Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans. J Biol Chem 273, 2607826086.
Lo, H. J., Kohler, J. R., DiDomenico, B., Loebenberg, D., Cacciapuoti, A. & Fink, G. R. (1997). Nonfilamentous C. albicans mutants are avirulent. Cell 90, 939-949.[Medline]
Merson-Davies, L. A. & Odds, F. C. (1989). A morphology index for characterization of cell shape in Candida albicans. J Gen Microbiol 135, 3143-3152.[Medline]
Mio, T., Yabe, T., Sudoh, M., Satoh, Y., Nakajima, T., Arisawa, M. & Yamada-Okabe, H. (1996). Role of three chitin synthase genes in the growth of Candida albicans. J Bacteriol 178, 2416-2419.[Abstract]
Monge, R. A., Navarro-Garcia, F., Molero, G., Diez-Orejas, R., Gustin, M., Pla, J., Sanchez, M. & Nombela, C. (1999). Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J Bacteriol 181, 3058-3068.
Odds, F. C. (1988). Morphogenesis in Candida, with special reference to C. albicans. In Candida and Candidosis, pp. 4259. London: Baillière Tindall.
Odds, F. C. (1991). Quantitative microculture system with standardized inocula for strain typing, susceptibility testing, and other physiologic measurements with Candida albicans and other yeasts. J Clin Microbiol 29, 2735-2740.[Medline]
Odds, F. C., Cockayne, A., Hayward, J. & Abbott, A. B. (1985). Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae. J Gen Microbiol 131, 2581-2589.[Medline]
Rieg, G., Fu, Y., Ibrahim, A. S., Zhou, X., Filler, S. G. & Edwards, J. E.Jr (1999). Unanticipated heterogeneity in growth rate and virulence among Candida albicans AAF1 null mutants. Infect Immun 67, 3193-3198.
Rink, R. D., Kaelin, C. R. & Fry, D. E. (1981). Lethal candidiasis in the rat: effects on metabolism and hepatic oxygen supply. J Surg Res 30, 75-79.[Medline]
Sanglard, D., Hube, B., Monod, M., Odds, F. C. & Gow, N. A. R. (1997). A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect Immun 65, 3539-3546.[Abstract]
Sarthy, A. V., McGonigal, T., Coen, D., Frost, D. J., Meulbroek, J. A. & Goldman, R. C. (1997). Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-ß-glucosyltransferase. Microbiology 143, 367-376.[Abstract]
Sawyer, R. T., Moon, R. J. & Beneke, E. S. (1976). Hepatic clearance of Candida albicans in rats. Infect Immun 14, 1348-1355.[Medline]
Timpel, C., Strahl-Bolsinger, S., Ziegelbauer, K. & Ernst, J. F. (1998). Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans. J Biol Chem 273, 20837-20846.
Winblad, B. (1975). Experimental renal candidiasis in mice and guinea pigs. Acta Pathol Microbiol Scand A 83, 406-414.[Medline]
Wysong, D. R., Christin, L., Sugar, A. M., Robbins, P. W. & Diamond, R. D. (1998). Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene. Infect Immun 66, 1953-1961.
Yaar, L., Mevarech, M. & Koltin, Y. (1997). A Candida albicans RAS-related gene (CaRSR1) is involved in budding, cell morphogenesis and hypha development. Microbiology 143, 3033-3044.[Abstract]
Yamada-Okabe, T., Mio, T., Ono, N., Kashima, Y., Matsui, M., Arisawa, M. & Yamada-Okabe, H. (1999). Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans. J Bacteriol 181, 7243-7247.
Zhao, X. J., McElhaney-Feser, G. E., Sheridan, M. J., Broedel, S. E.Jr & Cihlar, R. L. (1997). Avirulence of Candida albicans FAS2 mutants in a mouse model of systemic candidiasis. Infect Immun 65, 829-832.[Abstract]
Received 1 December 1999;
revised 2 May 2000;
accepted 17 May 2000.