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Infection and Immunity, October 2005, p. 7047-7050, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.7047-7050.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Relationship of Virulence Factor Expression to Evolved Virulence in Mouse-Passaged Cryptococcus neoformans Lines

Erin E. McClelland,1* Wesley T. Perrine,2 Wayne K. Potts,2 and Arturo Casadevall1

Department of Medicine, 702 Golding, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461,1 Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, Utah 841122

Received 11 March 2005/ Returned for modification 25 April 2005/ Accepted 27 May 2005


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ABSTRACT
 
Serial passage of Cryptococcus neoformans in mice increases virulence relative to the nonpassaged line. Postpassaged lines showed no difference in the expression of most known virulence factors, with the exception that the more virulent lines had smaller capsules in vitro. These data imply that other mechanisms of virulence remain to be discovered.


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TEXT
 
One approach to study the evolution of virulence experimentally is to serially passage a microbe in a host (8) and then compare the pre- and postpassaged lines to identify traits that affect changes in virulence. The yeast Cryptococcus neoformans is commonly found in the environment and causes disease primarily in immunocompromised humans. Its best-recognized virulence factors are the capsule (1, 14), melanin production (15, 25), laccase (17, 22), phospholipase (5), urease (6), and growth rate (18). Passage of C. neoformans in B10 major histocompatibility complex-congenic mice and BALB/c mice increased virulence relative to the nonpassaged line, using time to death as the measure of virulence (19). These observations prompted this study to investigate the mechanism responsible for the virulence increase.

The C. neoformans lines used in this study were derived by serial passage in mice and are described in detail in reference 19. Capsule size in vitro was measured as described in reference 26. Capsule size in vivo was measured from frozen brain and liver homogenates scraped into a microcentrifuge tube, washed once with 100 µl phosphate-buffered saline, resuspended in 10 µl phosphate-buffered saline, and measured as described in reference 26. To determine if the lines differed in their ability to release capsular glucuronoxylomannan (GXM) into the medium, capsules were induced in DME (as for measuring capsule size) and the concentration of GXM in the supernatant was measured the next day by capture enzyme-linked immunosorbent assay as described previously (2).

Melanization was assessed qualitatively by colony color on L-dopa plates after incubation at 30°C for 3, 5, and 7 days. The amount of color produced was scored based on colony photographs using a 0 to 5 scale, with 0 being white and 5 corresponding to black. Laccase activity was measured as described previously (13). Extracellular phospholipase activity was determined as described previously (9). Urease activity was determined as described previously (16).

All lines were grown in yeast-peptone-dextrose medium at 37°C overnight and then diluted in 10 ml yeast-peptone-dextrose medium and the growth rate measured as described previously (10) from CFU and turbidity. CFU were measured three times (every 4 h). The doubling time was calculated using the following formula: time x {0.693/[ln(final OD/initial OD)]}, in which OD is the optical density.

The phagocytic efficacy of the macrophage-like cell line J774.16 was measured by the method used in reference 24, with minor modifications. Macrophage killing was measured by the method used in reference 12, with modifications. Briefly, J774.16 cells were opsonized with either 10% guinea pig serum or 10 µg/ml of the monoclonal antibody 18B7. Postpassaged lines and macrophages were incubated in a 1:1 ratio for 4- and 18-h intervals, and viability was assessed by trypan blue exclusion.

A logistic correlation was used to test if virulence factors were correlated with time to death (virulence) during the last passage. A standard least-squares test with simple contrasts was used to test for significant differences between nonpassaged and postpassaged C. neoformans macrophage killing.

Surprisingly, there was no correlation between time to death and the following line characteristics: capsule GXM release in vitro, secreted extracellular laccase, extracellular phospholipase secretion, urease activity, growth rates, and in vitro phagocytosis with mouse intraperitoneal macrophages (Table 1). There was also no significant difference in macrophage killing between the nonpassaged H99 and any postpassaged line (Table 1). Furthermore, there was no correlation between the amount of melanization and the time to death (Table 1). Eight of the 14 C. neoformans passaged lines produced some melanin by day 7, but only 5 of those 8 produced a significant amount of melanin by day 7 (Table 1). There was no correlation between capsule size in the brain or liver of moribund mice and passage time to death (Table 1). However, a negative correlation between capsule size in vitro and passage time to death (virulence) was found (P = 0.0347, R2 = 0.28) (Table 1). All but one (F1) of the passaged C. neoformans lines produced a capsule smaller than the capsule of the nonpassaged H99 (Fig. 1) in vitro.


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  		TABLE 1. Correlation analysis for virulence factors and C. neoformans strains



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  		FIG. 1. Correlation between capsule size and passage time to death. Points are the average in vitro capsule size of each line plotted against passage time to death. Strain names indicate the MHC genotype or strain of mice in which C. neoformans was passaged (19).

There was a statistically significant negative correlation between increased virulence (decreased time to death) and smaller capsule size in vitro (P = 0.0347) that is opposite that of conventional views on capsule size and virulence. This observation, combined with the finding that mice infected with passaged lines had higher GXM serum levels (data not shown), suggests that increased virulence was due either to more in vivo capsule shedding or to an increased growth rate in vivo. Since soluble capsular polysaccharide can mediate many deleterious effects on the immune system, including alteration in cytokine regulation (21, 23), interference with leukocyte migration (7), and apoptosis (4), the finding of smaller capsules and increased serum polysaccharide suggests a potential explanation in addition to increased growth rate in vivo, for the increased virulence of the highly virulent mouse-passaged lines.

Growth rate/doubling time was tested in vitro in different media, but no correlation with time to death was found. We did not measure growth rate in vivo because that would require separating differences in replication rate from changes in tissue burden as a result of clearance by immune cells. Since increased growth rate in vivo may still be a possible mechanism for the increased virulence seen in these mouse-passaged lines of C. neoformans, we cannot exclude this mechanism.

From a microbe-centric perspective, there are several potential explanations for these observations. First, the virulence factors that remained unchanged in postpassage lines may function in a qualitative manner such that only their presence is required for virulence. The fact that quantitative differences in virulence factor expression have not been associated with virulence in C. neoformans supports this view. Second, mouse passage may not affect essential fungal characteristics that arose in the environment and function as virulence factors in mammals. In this regard, we note that the capsule of C. neoformans seldom elicits high-titer or protective antibody responses in natural infections (3, 20), and consequently, this trait may not be under strong immune selection pressure. Similarly, murine infection does not elicit antibodies to laccase (11). Third, line adaptation to survival in mice may involve selection for other virulence factors.

In conclusion, mouse passage can increase the virulence of C. neoformans without selecting for significant differences in many well-characterized virulence factors. The increased virulence seen in the highly virulent lines may be due to a combination of increased growth rate in vivo, increased serum capsular polysaccharide, or changes in undiscovered virulence factors.


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ACKNOWLEDGMENTS
 
We thank Javier Garcia-Rivera for help with the melanization and laccase experiments, Luis Martinez for help with GXM enzyme-linked immunosorbent assays, Oscar Zaragoza for help with measuring capsule and cell sizes, Emily Cook for general technical help, Megan McClelland for statistical help, and Diane McFadden and Helene Eisenman for critical discussion.

This study was in part supported by NIH grant GM-39578 to W.K.P. and NIH grants GM-071421, AI033142, AI033774, AI052733, and HL059842 to A.C.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine, 702 Golding, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-3768. Fax: (718) 430-8701. E-mail: mcclella{at}aecom.yu.edu. Back

Editor: T. R. Kozel


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Infection and Immunity, October 2005, p. 7047-7050, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.7047-7050.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • McClelland, E. E., Bernhardt, P., Casadevall, A. (2006). Estimating the Relative Contributions of Virulence Factors for Pathogenic Microbes. Infect. Immun. 74: 1500-1504 [Abstract] [Full Text]  

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