MYCOSES
ACCEPTED: JANUARY 15, 1992
35, 9-16 (1992)
Virulence of Candida albicans mutants
Virulenz von Candida albicans-Mutanten Annemarie Polak Key words. Candida albicans, mutant strains, virulence, animal model. Schliisselworter. Candida albicans, Mutanten, Virulenz, Tiermodell.
Summary. Mutant strains of the fungal pathogen Cundida albicuns blocked in pyrimidine transport and salvage metabolism were tested for virulence in various animal models. The growth rate, germination and proteolytic enzyme production did not correlate with the virulence of the strains. However, a defect in the uridine transport system significantly decreased virulence in murine candidosis, although it had no effect in vaginal candidosis or in a Cundida cyst model. It remains unclear whether this is due to the differing host defence mechanisms involved in systemic and superficial mycoses, or to the different requirements of the fungal systems for adherence and tissue invasion in the two types of infection. Zusammenfassung. Mutanten des pathogenen Hefepilzes Cundida ulbicuns mit blockiertem Pyrimidintransport und Abbaustoffwechsel wurden in verschiedenen Tiermodellen auf Virulenzunterschiede gepruft. Die Wachstumsrate, die Auskeimung und die Aktivitat proteolytischer Enzyme korrelierte nicht mit der Virulenz der Stamme. Hingegen verminderte ein Defekt im Uridintransportsystem signifikant die Virulenz im Model1 der systemischen murinen Candidose, obwohl dieser Defekt andererseits keine Wirkung auf die Vaginalcandidose und auf das CundidaHautzystenmodell hatte. Es bleibt offen, ob dies in unterschiedlichen Wirtsabwehrmechanismen bei systemischen und oberflachlichen Mykosen oder aber in unterschiedlichen Adharenz- und F. Hoffmann-La Roche Ltd, Pharma Division, Pharmaceutical Research, Basel, Switzerland. Correspondence: PD Dr Annemarie Polak, F. HoffmannLa Roche Ltd, Grenzacherstr. 124, CH-4002 Basel, Switzerland.
Gewebsinvasionsbedingungen auf seiten des pilzlichen Erregers bei den beiden Mykoseformen begriindet ist.
Introduction
Candida ulbicuns is the most common etiological agent of candidosis [ 13. Its pathological manifestations range from vaginitis thrush to systemic mycosis and it is particularly important in immunosuppressed patients. Effective systemic antifungals are still few in number and new approaches for influencing Candida infections are actively being sought. One possible new approach involves the host directly rather than the infecting agent-stimulation of reduced immunodefence by immune modulators. Another alternative is to attack target enzymes whose absence or inhibition, although not lethal for the fungus, leads to a loss of virulence. Some virulence factors of fungi are well established. The virulence of C. albicans strains has been found to be strongly dependent on phospholipase and proteinase production [2-51, but other factors may also influence the pathogenicity of yeasts. Sterol biosynthesis inhibitors significantly reduce the virulence of C. albicuns strains [6, Polak, unpublished], but the mechanism of this effect is not known. Certain auxotrophic C. albicuns mutants showed a lowered level of pathogenicity relative to control wild strains, but again no explanation at the molecular level is apparent [7]. However, these observations suggest that metabolic abnormalities can lead to a reduction in virulence. A particularly well-characterized set of C. albicans mutants was produced by M. Fasioli
10
A. POLAK
[8, 91, who studied in detail their genetic and biochemical behaviour. These strains all have altered pyrimidine transport and salvage metabolism which leads to a variation of their sensitivity to the antifungal 5-flucytosine (5-FC). Fasioli also reported a lack of pathogenicity in these mutants
POI.
We have now studied seven of Fasioli's mutants and their wild type parent together with two of our laboratory wild type strains with respect to their various microbiological properties in an attempt to correlate these with each other and with the pathogenicity of the strains in animal models. It would be of great clinical relevance if these specific mutations in a natural transport and salvage system which alter drug susceptibility did in fact lead to a reduced virulence.
Materials and methods Candida albicans strains The strains used in this study are listed in Table 1 together with the mutations they contain. Strain MEN is the wild type parent of the seven mutants, and H12 and H29 are the virulent wild type strains routinely used in our animal models of Candida infections. Estimation of generation time The strains were grown overnight at 37°C in Yeast Nitrogen Base and then diluted and resuspended in the same medium at an initial inoculum of 1 x lo6 yeast cellslml. Growth was followed by haemocytometer count over a period of 8 h and the generation time calculated. Extent of germination Strains were grown overnight at 37 "C on Sabouraud glucose agar, harvested and resuspended in human serum at 1 x lo6 yeast cells/ml. Over a period of 6-8 h the percentage of germinating cells was estimated. Determination of phospholipase production Generally, the method as described by Price et al. [ l l ] was used. The test medium consisted of Sabouraud agar containing 1 M sodium chloride, 0.005 M calcium chloride and 2% egg yolk (bacto egg yolk enrichment 5oyO, 4 ml in 100 ml agar). Small Petri dishes were filled with 4.5ml agar+0.5 ml water, and 10 p1 of a thick cell suspension of the C. albicans strain was placed in
the centre of the plate after the agar had set. Measurement and calculation of the zone of phospholipase activity (PZ) was performed according to the method described by Price et al. [ l 11. The test medium is initially translucent and a wide dense zone of precipitation around the colonies of phospholipase-producing C. albicans was distinct and well defined. Phospholipase activity was measured in terms of the ratio of the diameter of the colony to the total diameter of colony zone of precipitation. The lower this PZ value, the higher the production of phospholipase activity .
+
Proteinase activity The extent of proteinase secretion was estimated on agar plates containing BSA prepared according to Staib [12]. Five-centimetre plates were prepared with 1% Agar Noble (Difco) containing 0.1 yo KH,PO,, 0.05% MgSO,, 1yo sucrose and 0.16y0 BSA. C. albicans strains were preincubated for 24 h in Sabouraud medium and the centrifuged cells washed twice in sterile physiological saline. Ten microlitres of a suspension in a minimal amount of saline was then carefully added to the centre of the agar plate. After incubation at 37°C for 5 days the plates were stained with amido black and destained with 7% aqueous acetic acid. The diameter of the unstained zone around the colony is a measure of proteinase production. Animal models Murine candidosis. The mice used for these experiments were male albino Fullinsdorf mice weighing approximately 20 g. Groups of 5 or 10 mice were infected by lateral tail vein injection of 0.2 ml of C. albicans cell suspension using various inoculum sizes. The mice were observed for a minimum of 20 days, with death recorded on a daily basis. At the end of the observation time an LD,, and an LD,, were calculated, i.e. the inoculum resulting in the death of 50% and 90% of the animals. Additionally, the median survival time was calculated at a n inoculum of 1 x lo6 Candida cells. In a second experiment the mice were treated with cyclophosphamide on day 3 before inoculation. The animals became neutropenic and the LD and LD,, inocula were significantly 5? lower than in normal, immunocompetent mice. Again, as well as the LD50 and LD,,, the median survival time was calculated. Vaginal candidosis in the rat. SPF albino Fullinsdorf rats were used in this model. Females weighing mycoses 35, 9- 16 ( 1992)
VIRULENCE OF C. ALBICANS MUTANTS
approximately 40 g were ovarectomized, kept in a state of permanent oestrus by injecting oestradiol and infected intravaginally by instillation of approximately 5 x lo6 yeast cells from a fresh 48 h subculture (Sabouraud 37 "C) ofthe relevant strain. This infection method resulted consistently in vaginal candidosis which was fully developed 24 h later. The colony forming units (CFU) in the vagina were measured at intervals of 24 h, 3 days and 7 days after infection. Candida cyst model. A model of localized candidosis resulting in thrush-like lesions in artificial subcutaneous cysts in mice has recently been developed [ 131. Albino mice were injected subcutaneously with 2-3 ml of air through a hypodermic needle. Every 3 to 4 days air loss by diffusion from the cyst was replaced, but without increasing the initial size of the cyst. Five to 10 days after establishment of the cyst the animals were subjected to immunosuppressive therapy with cyclophosphamide and 3 days later challenged subcutaneously directly into the cyst with lo6 yeast cells suspended in 0.1 ml of saline. The course of the infection was then observed at various time intervals up to 1 1 days after infection by excising the cysts and homogenizing them in 10 ml of saline. The colony forming units in this suspension were determined using a 1 : 10 dilution series on Sabouraud glucose agar containing antibiotics. The colonies of c. albicans cells were read after an incubation period of 2 days at 37 "C.
Results In vitro characterization of Candida albicans strains The strains studied and their genetic and microbiological properties are shown in Table 1. Three strains can be considered as wild type in that they are fully 5-FC sensitive. These are the two reference strains H12 and H29, and strain 72s. This latter was in fact selected from Fasioli's parent strain MEN and shown to be homozygous for UMP pyrophosphorylase, whereas the MEN itself is heterozygous for this gene and thus partially 5-FC resistant. Strain 72R lacks the gene completely and is thus fully resistant. Strain GF1, a mutant of 72S, lacks the uridine permease, but this has no effect on 5-FC sensitivity. Four double mutants were produced from GFl with the mutations indicated in Table 1. All are resistant to 5-FC. As can be seen from Table 1 there is no correlation between 5-FC resistance and cell growth. All strains grow normally on a chemically defined mycoses 35, 9- 16 ( 1992)
11
medium (YNB). In all strains germ formation is induced in human serum. The degree of germination again varies significantly from strain to strain, the highest being seen in strain GFl (53% at 5 h), the lowest in the mutant strain F1 (5.5%). Our own laboratory strain H29 also germinates poorly, with only 8.4% germination at 5 h. However, despite these considerable variations there is no correlation between 5-FC sensitivity and degree of germination. All 10 strains have phospholipase activity, production of which is one of the known virulence factors of C. albicans. Phospholipase activity is similar in all strains except E l , which produces significantly more than the others. Again there is no distinction between 5-FC mutated strains and wild strains with regard to phospholipase production. The same is true for proteinase production, another known virulence factor. Virulence in murine candidosis All 9 strains examined induced Candida infection in both immunocompetent and neutropenic mice. The mean survival time and the inoculum values for 50% and 90% mortality are shown in Table 2, and the main feature is that some strains have a steep dose-activity curve (Fig. I ) , while for others it is very flat. This characteristic is not correlated with 5-FC sensitivity, but is found in both wild and mutant strains. When the LD,, and LD,, values and the mean survival time are studied, two distinctive groups of strains can be observed. H12, MEM, 72s and 72R cause acute disease with short survival time, and a low inoculum is needed to induce a 50% or 90% mortality. All strains with a mutation at the locus of uridine permease, i.e. the strain GFI with a single mutation and the strains E l , E2, F1, F2 with the double mutation, belong to a second group, characterized' by a significantly longer mean survival time at an inoculum of 1 x lo6 yeast cells/mouse, i.e. a less acute disease. Higher numbers of yeast cells are necessary to induce a 50% or 90% mortality. This difference is seen in immunocompetent as well as in neutropenic mice. The difference between the two groups is more marked in neutropenic mice, where a lower amount (at least one log lower) of inoculum is generally necessary to induce an acute candidosis, the course of infection is significantly more rapid, and the mice succumb to the disease earlier than the immunocompetent mice. This can be seen from the low mean survival time of 1.2 to 5.3 days at an inoculum of lo5 yeast cells in group 1 as well as the yeast load required to induce a 50% mor-
Table 1. Characteristics of the 8 C. albicans strains with specific mutational changes and of two wild strains HI2 and H29 Generation time (min)
Germination at 5 h in human serum (in yo)
Phospholipase production3
Proteinase produc tion§
65 66 50
70 80 95
31.6 8.4 10.8
0.56 0.56 0.53
20 20 19.5
33
64
90
12.3
0.61
21.5
0
0
78
15.0
0.57
17.5
32 0 0 0 0
62 0 0 0 0
66 78 a4 96 78
53.0 15.7 20.1 5.5 9.8
0.59 0.49 0.55 0.56 0.55
20.5 21.5 17.5 17.5 19
Strain
Parental strain
Phenotype?
5-FC sensitivity*
H12
Clin. isolate Lab. strain
WILD WILD Heterozygous for UMP pyr Homozygous wild for UMP pyr+ Homozygous for UMP pyrUrd perUrd perCyt deamUrd perUMP pyr-
32 31 0
H29 MEN
72s
MEN
72R
MEN
GF 1 El E2
72s GF 1 GF 1 GF 1 GFI
F1 F2
} }
*5-FC sensitivity quoted as diffusion 4 (mm) using 1 and 100 pg 5-FC disks.
t UMP pyr, UMP pyrophosphorylase; URD per, uridine permease; Cyt deam, cytosine dearninase. IPZ values calculated according to Price cf a f . [ 1 I]. §Zone of lysis.
VIRULENCE OF C. ALBICANS MUTANTS
13
Table 2. Murine candidosis in normal and neutropenic mice Strain
Normal mice
Neutropenic mice
MST
LD50
LD90
MST
LD50
LD90
H12 MEN 72s 72R
5.9 4.6 6.8 6.8
4.5 4.7 4.6 5.18
4.88 5.23 5.85 5.78
1.2 4.4 5.3 2.6
3.08 3.58 3.98 3.40
3.5 4.08 4.38 4.54
Group 1
GF 1 El E2 F1 F2
12.7 15.7 17.4 15.2 12.2
5.81 5.62 5.95 5.81 5.7
6.48 6.65 6.70 6.95 6.0
16.2 18.2 14.2 15.7 13.3
5.15 5.45 4.74 5.08 5.0
5.62 6.30 5.70 5.48 5.60
Group 2
MST: values represent mean survival time with 1 x lo6 in normal mice and 1 x lo5 in neutropenic mice. LD,,: values expressed as log number of yeast cells that resulted in a 50% mortality at day 20. LD,: values expressed as log number of yeast cells that resulted in a 90% mortality at day 20.
100-
80-
-0
Table 3. cyst
Localized candidosis. Candida cells (CFU) per
Strain
Days after infection
VI
.c
60-
Day 4
Day 7*t
Day 11
1 x 1072.74 x los** 1.02 x 106 3.1 x 105** 7.5 x 105** 1 . o x lo6 1.3 x 106 1 . 8 lo6 ~ 1 . 1 3 lo6 ~
3.2 x 107 1.37 x lo6** 5.8 x 105 3.66 x 105 1.08 x lo6* 1.58 x lo6** 6.8 x lo5 8.12 x 105 8 x lo5
8 x lo6 3.9 x 105 7.5 x 105 1.48 x 105. 9.9 x 105 5.3 x 105 7.3 x 105 7.9 x 105 1.2 x 106
~~
0
-0
40-
U
x 20-
0 I
lnoculurn per mouse
Figure 1. Inoculum effect in dead animals 20 days after infection of various amounts of C. albicans.
tality. The mean survival time in group 2 is significantly longer at the inoculum of lo5 than in group 1 and a high inoculum is necessary for a 50% mortality. In some cases there is no difference between immunocompetent and neutropenic mice as far as the inoculum size of group 2 strains is concerned. So a clear difference in virulence is seen in murine candidosis between strains having uridine permease activity (H12, MEM, 72s and 72R) and those lacking this gene (GF1, E l , E2, F1 and F2).
Localized candidosis I n Table 3 and Figure 2 the course of infection in the localized candidosis model is shown. In this model the grouping observed in murine organ candidosis is not apparent. However, a significant difference is observed between our laboratory wild strain H29 and all other 8 strains studied. mycoses 35, 9-16 (1992)
H29 MEN 72s 72R GF I El E2 F1 F2
?On day 7 the plateau of CFU in cyst is reached. *Significant difference to CFU in 72s (P
ACCEPTED: JANUARY 15, 1992
35, 9-16 (1992)
Virulence of Candida albicans mutants
Virulenz von Candida albicans-Mutanten Annemarie Polak Key words. Candida albicans, mutant strains, virulence, animal model. Schliisselworter. Candida albicans, Mutanten, Virulenz, Tiermodell.
Summary. Mutant strains of the fungal pathogen Cundida albicuns blocked in pyrimidine transport and salvage metabolism were tested for virulence in various animal models. The growth rate, germination and proteolytic enzyme production did not correlate with the virulence of the strains. However, a defect in the uridine transport system significantly decreased virulence in murine candidosis, although it had no effect in vaginal candidosis or in a Cundida cyst model. It remains unclear whether this is due to the differing host defence mechanisms involved in systemic and superficial mycoses, or to the different requirements of the fungal systems for adherence and tissue invasion in the two types of infection. Zusammenfassung. Mutanten des pathogenen Hefepilzes Cundida ulbicuns mit blockiertem Pyrimidintransport und Abbaustoffwechsel wurden in verschiedenen Tiermodellen auf Virulenzunterschiede gepruft. Die Wachstumsrate, die Auskeimung und die Aktivitat proteolytischer Enzyme korrelierte nicht mit der Virulenz der Stamme. Hingegen verminderte ein Defekt im Uridintransportsystem signifikant die Virulenz im Model1 der systemischen murinen Candidose, obwohl dieser Defekt andererseits keine Wirkung auf die Vaginalcandidose und auf das CundidaHautzystenmodell hatte. Es bleibt offen, ob dies in unterschiedlichen Wirtsabwehrmechanismen bei systemischen und oberflachlichen Mykosen oder aber in unterschiedlichen Adharenz- und F. Hoffmann-La Roche Ltd, Pharma Division, Pharmaceutical Research, Basel, Switzerland. Correspondence: PD Dr Annemarie Polak, F. HoffmannLa Roche Ltd, Grenzacherstr. 124, CH-4002 Basel, Switzerland.
Gewebsinvasionsbedingungen auf seiten des pilzlichen Erregers bei den beiden Mykoseformen begriindet ist.
Introduction
Candida ulbicuns is the most common etiological agent of candidosis [ 13. Its pathological manifestations range from vaginitis thrush to systemic mycosis and it is particularly important in immunosuppressed patients. Effective systemic antifungals are still few in number and new approaches for influencing Candida infections are actively being sought. One possible new approach involves the host directly rather than the infecting agent-stimulation of reduced immunodefence by immune modulators. Another alternative is to attack target enzymes whose absence or inhibition, although not lethal for the fungus, leads to a loss of virulence. Some virulence factors of fungi are well established. The virulence of C. albicans strains has been found to be strongly dependent on phospholipase and proteinase production [2-51, but other factors may also influence the pathogenicity of yeasts. Sterol biosynthesis inhibitors significantly reduce the virulence of C. albicuns strains [6, Polak, unpublished], but the mechanism of this effect is not known. Certain auxotrophic C. albicuns mutants showed a lowered level of pathogenicity relative to control wild strains, but again no explanation at the molecular level is apparent [7]. However, these observations suggest that metabolic abnormalities can lead to a reduction in virulence. A particularly well-characterized set of C. albicans mutants was produced by M. Fasioli
10
A. POLAK
[8, 91, who studied in detail their genetic and biochemical behaviour. These strains all have altered pyrimidine transport and salvage metabolism which leads to a variation of their sensitivity to the antifungal 5-flucytosine (5-FC). Fasioli also reported a lack of pathogenicity in these mutants
POI.
We have now studied seven of Fasioli's mutants and their wild type parent together with two of our laboratory wild type strains with respect to their various microbiological properties in an attempt to correlate these with each other and with the pathogenicity of the strains in animal models. It would be of great clinical relevance if these specific mutations in a natural transport and salvage system which alter drug susceptibility did in fact lead to a reduced virulence.
Materials and methods Candida albicans strains The strains used in this study are listed in Table 1 together with the mutations they contain. Strain MEN is the wild type parent of the seven mutants, and H12 and H29 are the virulent wild type strains routinely used in our animal models of Candida infections. Estimation of generation time The strains were grown overnight at 37°C in Yeast Nitrogen Base and then diluted and resuspended in the same medium at an initial inoculum of 1 x lo6 yeast cellslml. Growth was followed by haemocytometer count over a period of 8 h and the generation time calculated. Extent of germination Strains were grown overnight at 37 "C on Sabouraud glucose agar, harvested and resuspended in human serum at 1 x lo6 yeast cells/ml. Over a period of 6-8 h the percentage of germinating cells was estimated. Determination of phospholipase production Generally, the method as described by Price et al. [ l l ] was used. The test medium consisted of Sabouraud agar containing 1 M sodium chloride, 0.005 M calcium chloride and 2% egg yolk (bacto egg yolk enrichment 5oyO, 4 ml in 100 ml agar). Small Petri dishes were filled with 4.5ml agar+0.5 ml water, and 10 p1 of a thick cell suspension of the C. albicans strain was placed in
the centre of the plate after the agar had set. Measurement and calculation of the zone of phospholipase activity (PZ) was performed according to the method described by Price et al. [ l 11. The test medium is initially translucent and a wide dense zone of precipitation around the colonies of phospholipase-producing C. albicans was distinct and well defined. Phospholipase activity was measured in terms of the ratio of the diameter of the colony to the total diameter of colony zone of precipitation. The lower this PZ value, the higher the production of phospholipase activity .
+
Proteinase activity The extent of proteinase secretion was estimated on agar plates containing BSA prepared according to Staib [12]. Five-centimetre plates were prepared with 1% Agar Noble (Difco) containing 0.1 yo KH,PO,, 0.05% MgSO,, 1yo sucrose and 0.16y0 BSA. C. albicans strains were preincubated for 24 h in Sabouraud medium and the centrifuged cells washed twice in sterile physiological saline. Ten microlitres of a suspension in a minimal amount of saline was then carefully added to the centre of the agar plate. After incubation at 37°C for 5 days the plates were stained with amido black and destained with 7% aqueous acetic acid. The diameter of the unstained zone around the colony is a measure of proteinase production. Animal models Murine candidosis. The mice used for these experiments were male albino Fullinsdorf mice weighing approximately 20 g. Groups of 5 or 10 mice were infected by lateral tail vein injection of 0.2 ml of C. albicans cell suspension using various inoculum sizes. The mice were observed for a minimum of 20 days, with death recorded on a daily basis. At the end of the observation time an LD,, and an LD,, were calculated, i.e. the inoculum resulting in the death of 50% and 90% of the animals. Additionally, the median survival time was calculated at a n inoculum of 1 x lo6 Candida cells. In a second experiment the mice were treated with cyclophosphamide on day 3 before inoculation. The animals became neutropenic and the LD and LD,, inocula were significantly 5? lower than in normal, immunocompetent mice. Again, as well as the LD50 and LD,,, the median survival time was calculated. Vaginal candidosis in the rat. SPF albino Fullinsdorf rats were used in this model. Females weighing mycoses 35, 9- 16 ( 1992)
VIRULENCE OF C. ALBICANS MUTANTS
approximately 40 g were ovarectomized, kept in a state of permanent oestrus by injecting oestradiol and infected intravaginally by instillation of approximately 5 x lo6 yeast cells from a fresh 48 h subculture (Sabouraud 37 "C) ofthe relevant strain. This infection method resulted consistently in vaginal candidosis which was fully developed 24 h later. The colony forming units (CFU) in the vagina were measured at intervals of 24 h, 3 days and 7 days after infection. Candida cyst model. A model of localized candidosis resulting in thrush-like lesions in artificial subcutaneous cysts in mice has recently been developed [ 131. Albino mice were injected subcutaneously with 2-3 ml of air through a hypodermic needle. Every 3 to 4 days air loss by diffusion from the cyst was replaced, but without increasing the initial size of the cyst. Five to 10 days after establishment of the cyst the animals were subjected to immunosuppressive therapy with cyclophosphamide and 3 days later challenged subcutaneously directly into the cyst with lo6 yeast cells suspended in 0.1 ml of saline. The course of the infection was then observed at various time intervals up to 1 1 days after infection by excising the cysts and homogenizing them in 10 ml of saline. The colony forming units in this suspension were determined using a 1 : 10 dilution series on Sabouraud glucose agar containing antibiotics. The colonies of c. albicans cells were read after an incubation period of 2 days at 37 "C.
Results In vitro characterization of Candida albicans strains The strains studied and their genetic and microbiological properties are shown in Table 1. Three strains can be considered as wild type in that they are fully 5-FC sensitive. These are the two reference strains H12 and H29, and strain 72s. This latter was in fact selected from Fasioli's parent strain MEN and shown to be homozygous for UMP pyrophosphorylase, whereas the MEN itself is heterozygous for this gene and thus partially 5-FC resistant. Strain 72R lacks the gene completely and is thus fully resistant. Strain GF1, a mutant of 72S, lacks the uridine permease, but this has no effect on 5-FC sensitivity. Four double mutants were produced from GFl with the mutations indicated in Table 1. All are resistant to 5-FC. As can be seen from Table 1 there is no correlation between 5-FC resistance and cell growth. All strains grow normally on a chemically defined mycoses 35, 9- 16 ( 1992)
11
medium (YNB). In all strains germ formation is induced in human serum. The degree of germination again varies significantly from strain to strain, the highest being seen in strain GFl (53% at 5 h), the lowest in the mutant strain F1 (5.5%). Our own laboratory strain H29 also germinates poorly, with only 8.4% germination at 5 h. However, despite these considerable variations there is no correlation between 5-FC sensitivity and degree of germination. All 10 strains have phospholipase activity, production of which is one of the known virulence factors of C. albicans. Phospholipase activity is similar in all strains except E l , which produces significantly more than the others. Again there is no distinction between 5-FC mutated strains and wild strains with regard to phospholipase production. The same is true for proteinase production, another known virulence factor. Virulence in murine candidosis All 9 strains examined induced Candida infection in both immunocompetent and neutropenic mice. The mean survival time and the inoculum values for 50% and 90% mortality are shown in Table 2, and the main feature is that some strains have a steep dose-activity curve (Fig. I ) , while for others it is very flat. This characteristic is not correlated with 5-FC sensitivity, but is found in both wild and mutant strains. When the LD,, and LD,, values and the mean survival time are studied, two distinctive groups of strains can be observed. H12, MEM, 72s and 72R cause acute disease with short survival time, and a low inoculum is needed to induce a 50% or 90% mortality. All strains with a mutation at the locus of uridine permease, i.e. the strain GFI with a single mutation and the strains E l , E2, F1, F2 with the double mutation, belong to a second group, characterized' by a significantly longer mean survival time at an inoculum of 1 x lo6 yeast cells/mouse, i.e. a less acute disease. Higher numbers of yeast cells are necessary to induce a 50% or 90% mortality. This difference is seen in immunocompetent as well as in neutropenic mice. The difference between the two groups is more marked in neutropenic mice, where a lower amount (at least one log lower) of inoculum is generally necessary to induce an acute candidosis, the course of infection is significantly more rapid, and the mice succumb to the disease earlier than the immunocompetent mice. This can be seen from the low mean survival time of 1.2 to 5.3 days at an inoculum of lo5 yeast cells in group 1 as well as the yeast load required to induce a 50% mor-
Table 1. Characteristics of the 8 C. albicans strains with specific mutational changes and of two wild strains HI2 and H29 Generation time (min)
Germination at 5 h in human serum (in yo)
Phospholipase production3
Proteinase produc tion§
65 66 50
70 80 95
31.6 8.4 10.8
0.56 0.56 0.53
20 20 19.5
33
64
90
12.3
0.61
21.5
0
0
78
15.0
0.57
17.5
32 0 0 0 0
62 0 0 0 0
66 78 a4 96 78
53.0 15.7 20.1 5.5 9.8
0.59 0.49 0.55 0.56 0.55
20.5 21.5 17.5 17.5 19
Strain
Parental strain
Phenotype?
5-FC sensitivity*
H12
Clin. isolate Lab. strain
WILD WILD Heterozygous for UMP pyr Homozygous wild for UMP pyr+ Homozygous for UMP pyrUrd perUrd perCyt deamUrd perUMP pyr-
32 31 0
H29 MEN
72s
MEN
72R
MEN
GF 1 El E2
72s GF 1 GF 1 GF 1 GFI
F1 F2
} }
*5-FC sensitivity quoted as diffusion 4 (mm) using 1 and 100 pg 5-FC disks.
t UMP pyr, UMP pyrophosphorylase; URD per, uridine permease; Cyt deam, cytosine dearninase. IPZ values calculated according to Price cf a f . [ 1 I]. §Zone of lysis.
VIRULENCE OF C. ALBICANS MUTANTS
13
Table 2. Murine candidosis in normal and neutropenic mice Strain
Normal mice
Neutropenic mice
MST
LD50
LD90
MST
LD50
LD90
H12 MEN 72s 72R
5.9 4.6 6.8 6.8
4.5 4.7 4.6 5.18
4.88 5.23 5.85 5.78
1.2 4.4 5.3 2.6
3.08 3.58 3.98 3.40
3.5 4.08 4.38 4.54
Group 1
GF 1 El E2 F1 F2
12.7 15.7 17.4 15.2 12.2
5.81 5.62 5.95 5.81 5.7
6.48 6.65 6.70 6.95 6.0
16.2 18.2 14.2 15.7 13.3
5.15 5.45 4.74 5.08 5.0
5.62 6.30 5.70 5.48 5.60
Group 2
MST: values represent mean survival time with 1 x lo6 in normal mice and 1 x lo5 in neutropenic mice. LD,,: values expressed as log number of yeast cells that resulted in a 50% mortality at day 20. LD,: values expressed as log number of yeast cells that resulted in a 90% mortality at day 20.
100-
80-
-0
Table 3. cyst
Localized candidosis. Candida cells (CFU) per
Strain
Days after infection
VI
.c
60-
Day 4
Day 7*t
Day 11
1 x 1072.74 x los** 1.02 x 106 3.1 x 105** 7.5 x 105** 1 . o x lo6 1.3 x 106 1 . 8 lo6 ~ 1 . 1 3 lo6 ~
3.2 x 107 1.37 x lo6** 5.8 x 105 3.66 x 105 1.08 x lo6* 1.58 x lo6** 6.8 x lo5 8.12 x 105 8 x lo5
8 x lo6 3.9 x 105 7.5 x 105 1.48 x 105. 9.9 x 105 5.3 x 105 7.3 x 105 7.9 x 105 1.2 x 106
~~
0
-0
40-
U
x 20-
0 I
lnoculurn per mouse
Figure 1. Inoculum effect in dead animals 20 days after infection of various amounts of C. albicans.
tality. The mean survival time in group 2 is significantly longer at the inoculum of lo5 than in group 1 and a high inoculum is necessary for a 50% mortality. In some cases there is no difference between immunocompetent and neutropenic mice as far as the inoculum size of group 2 strains is concerned. So a clear difference in virulence is seen in murine candidosis between strains having uridine permease activity (H12, MEM, 72s and 72R) and those lacking this gene (GF1, E l , E2, F1 and F2).
Localized candidosis I n Table 3 and Figure 2 the course of infection in the localized candidosis model is shown. In this model the grouping observed in murine organ candidosis is not apparent. However, a significant difference is observed between our laboratory wild strain H29 and all other 8 strains studied. mycoses 35, 9-16 (1992)
H29 MEN 72s 72R GF I El E2 F1 F2
?On day 7 the plateau of CFU in cyst is reached. *Significant difference to CFU in 72s (P
