In this study, we used a competitive infection model of hematogenously disseminated candidiasis in mice to redefine the role of C. albicans IRS4
in pathogenesis. Most notably, we demonstrated that IRS4
contributes to pathogenesis at the early stages of kidney invasion, rather than after several days, as suggested in our previous study (4
). Significant reductions in the tissue burdens of the Δirs4
mutant compared to CAI-12 (an isogenic, IRS4
-expressing strain) were evident within the kidneys at as early as 6 h after intravenous inoculation. Taken with our previous finding that the Δirs4
mutant adheres poorly to a variety of cell lines (4
), the data suggest that IRS4
is engaged in the pathogenic process from the initial contact of C. albicans
with the kidney. The attenuated virulence of the Δirs4
mutant did not reflect a generalized loss of fitness, as the mutant was not outcompeted by CAI-12 during routine growth in vitro
. Moreover, IRS4
does not appear to play a role during the hematogenous stage of disseminated disease, as gene expression by the mutant was not significantly altered within blood.
We also showed that IRS4 makes an additional, previously unrecognized contribution to later stages of kidney invasion. By day 4 of disseminated candidiasis, the burdens of the Δirs4 mutant within the kidneys were significantly lower during mixed infections with CAI-12 than mutant-only infections. This finding was in contrast to that at day 1, at which point the burdens of the Δirs4 mutant were similar in the presence or absence of CAI-12. Histopathology of infected kidneys over the first 4 days revealed significantly greater infiltration of PMNs and mononuclear cells, the front-line effectors of the innate immune system, in response to mixed infections than to mutant-only infections. Along these lines, the Δirs4 mutant was significantly more susceptible than CAI-12 to phagocytosis and killing by human PMNs and mouse macrophages in vitro. Therefore, data from the competitive infection model suggest that IRS4 makes distinct early and later contributions to virulence. The early contributions likely reflect roles in processes like adherence, tissue penetration, and progressive hyphal formation. The later contributions are likely to also include effects in mediating resistance to phagocytosis as disseminated candidiasis progresses.
It is certainly plausible that the cellular processes to which IRS4
contributes are important for tissue invasion and resistance to clearance by the host (4
). We have shown that disruption of IRS4
results in increased levels of PI(4,5)P2 (but not other phosphoinositides) and focal accumulations of excess PI(4,5)P2, septins, and cell wall protein within plasma membrane and cell wall invaginations (4
). These abnormalities are associated with profound derangements in cell wall integrity, as evidenced by hypersusceptibility to caspofungin and other cell wall stressors and impaired invasive growth (4
). Although we found that only 0.2% of genes were differentially expressed by the Δirs4
mutant in blood, it is notable that several downregulated genes are involved in cell wall-related processes such as UDP-N
-acetylglucosamine biosynthesis, septin regulation, and invasive growth. Independent of its essential role in maintaining cellular viability, the cell wall is central to the pathogenesis of invasive candidiasis (27
). It is the interface of the pathogen-host interaction and makes complex contributions to adherence, morphogenesis, and resistance to host defenses. Therefore, it is reasonable that a cell wall-defective mutant would be less able to penetrate or proliferate within target organs, which present ongoing physical and environmental stresses. Along these lines, our data indicate that C. albicans
is perceived to be less threatening by the host in the face of IRS4
disruption, as normally protective phagocytic infiltration and inflammatory responses within the kidneys are dampened. At the same time, the hypersusceptibility of the Δirs4
mutant to phagocytic killing is in keeping with impaired cell wall integrity.
The study highlights the major advantages of a competitive infection model over conventional models. First, competitive infections are more sensitive at detecting small differences in virulence between strains. In fact, the absolute differences in tissue burdens between the Δirs4
mutant and strain CAI-12 at 6 h were extremely small (median, ~2-fold). Nevertheless, the competitive model achieved robust statistical significance because the mutant was outcompeted by CAI-12 in each mouse. The sensitivity was heightened by the use of qPCR to quantitate GE rather than reliance upon colony-counting methods. The numbers of GE were approximately 1 log unit higher than the numbers of CFU, which likely reflects the ability of qPCR to more accurately detect higher fungal burdens associated with filamentous morphologies (18
). Even in single-strain infections, qPCR revealed statistically significant differences between the Δirs4
mutant and CAI-12 at 24 h that were not evident by CFU enumeration. A second advantage of competitive models is that they can be employed in conjunction with single-strain infections to study how specific factors may impact virulence by altering the interaction with the host immune system. For example, it was apparent from our single-strain infections that IRS4
induced rapid inflammatory responses. Nevertheless, the competitive model demonstrated that the attenuated virulence of the Δirs4
mutant above and beyond that seen in the single-strain infection was correlated with phagocyte infiltration and susceptibility to phagocytosis.
We conducted the competitive and single-strain infections using 8 to 10 mice at each time point, which was consistent with the design of our previous studies. It is important to realize, however, that competitive infections have the potential to spare large numbers of animals. To highlight the power of competitive infections in sparing animals while detecting relatively small differences in virulence between strains, consider an example in which the goal is to detect a 0.6-log-unit (~4-fold) difference in mean tissue burdens with 80% power, assuming a standard deviation of 0.55. In this scenario, 15 animals and one strain per time point would be needed in single-strain infections to detect significant differences in virulence. Therefore, 120 animals would be needed to compare two strains at four time points. In a competitive model with the same assumptions and a correlation coefficient of 0.8, only 5 animals and two strains per time point (or 20 animals to compare two strains at four time points) would be needed. In other words, similar conclusions could be reached using 17% (20/120) of the number of animals. Even if additional animals were used for histopathology or other studies of host responses, the net reduction in lives expended would be substantial.
In conclusion, our data suggest that competitive infection models should be routinely employed in the study of C. albicans virulence. In addition to their superior performance, they are technically straightforward and generate reproducible data. As we demonstrate, competitive infections are particularly useful for studying genes whose contributions to virulence may be subtle, temporally regulated, or influenced by interaction with the host immune system.