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. 2016 Mar;16(2):fow011.
doi: 10.1093/femsyr/fow011. Epub 2016 Feb 5.

Examination of the pathogenic potential of Candida albicans filamentous cells in an animal model of haematogenously disseminated candidiasis

Ian A Cleary  1 , Sara M Reinhard  2 , Anna L Lazzell  2 , Carlos Monteagudo  3 , Derek P Thomas  4 , Jose L Lopez-Ribot  2 , Stephen P Saville  5
Affiliations

Examination of the pathogenic potential of Candida albicans filamentous cells in an animal model of haematogenously disseminated candidiasis

Ian A Cleary et al. FEMS Yeast Res. 2016 Mar.

Abstract

The opportunistic fungal pathogen Candida albicans is an increasingly common threat to human health. Candida albicans grows in several morphologies and mutant strains locked in yeast or filamentous forms have attenuated virulence in the murine model of disseminated candidiasis. Thus, the ability to change shape is important for virulence. The transcriptional repressors Nrg1p and Tup1p are required for normal regulation of C. albicans morphology. Strains lacking either NRG1 or TUP1 are constitutively pseudohyphal under yeast growth conditions, and display attenuated virulence in the disseminated model. To dissect the relative importance of hyphae and pseudohyphae during an infection, we used strains in which the morphological transition could be externally manipulated through controlled expression of NRG1 or TUP1. Remarkably, hyphal form inocula retain the capacity to cause disease. Whilst induction of a pseudohyphal morphology through depletion of TUP1 did result in attenuated virulence, this was not due to a defect in the ability to escape the bloodstream. Instead, we observed that pseudohyphal cells are cleared from tissues much more efficiently than either hyphal (virulent) or yeast form (avirulent) cells, indicating that different C. albicans morphologies have distinct interactions with host cells during an infection.

Keywords: Candida albicans; NRG1; TUP1; filamentation; pseudohyphae; virulence.

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Figures

Graphical Abstract Figure.
Graphical Abstract Figure.
Different morphological forms of Candida albicans are similar in the ability to escape the bloodstream but pseudohyphae are cleared more efficiently.
Figure 1.
Figure 1.
Infections with hyphae are virulent. (A) The tet-NRG1 strain (SSY50-B) was induced to form hyphae (90 minutes, 37°C, YPD+DOX) and approximately 9.5 × 105 CFU injected into mice. Cell morphology of the infecting inoculum is illustrated in the inset panel. Virulence was attenuated when the infecting cells were subsequently directed to yeast form growth (No DOX group). (B) Approximately 5.7 × 105 CFU preformed hyphae (90 minutes, 37°C, YPD + serum) of a wild-type strain (CAF2-1) were injected into mice. Cell morphology of the inoculum is again illustrated in the inset panel. In both cases, preformed hyphae were able to cause disease in mice.
Figure 2.
Figure 2.
Abolishing TUP1 transcription from a regulatable promoter induces pseudohyphae. (A) When TUP1 is expressed (No DOX) our tet-TUP1 strain forms yeast under yeast growth conditions (YPD, 28°C) and hyphae under inducing conditions (RPMI-1640, 37°C). When grown in the presence of DOX, the strain forms pseudohyphae in both yeast and hypha inducing conditions. (B) Quantitative real-time PCR analysis was used to verify that expression of TUP1 from the tet-regulated allele was modulated by addition of doxycycline to the medium and that 3 h growth (YPD, 28°C) was sufficient to reduce TUP1 expression below the detectable level. Levels were normalized by ACT1 and expressed relative to the time zero sample (therefore those results are 1.0). Error bars represent the standard error of the mean. (C) After growth in the presence of doxycycline (YPD, 28°C) for 3 h chains and clumps of elongated pseudohyphal cells form. Cells grown in the absence of doxycycline continued to grow as isolated yeast form cells under the same conditions.
Figure 3.
Figure 3.
TUP1 transcript depletion attenuates virulence. (A) Mice succumbed to infection with the tet-TUP1 strain injected as yeast (approximately 5 × 105 CFU) and allowed to form hyphae like a wild-type strain (No DOX, dark lines). However, abolition of TUP1 transcription upon infection (Plus DOX, light lines) resulted in all of the mice surviving. (B) When cells were injected in the pseudohyphal form (after TUP1 depletion, approximately 5.3 × 105 CFU), virulence was attenuated, even when cells were able to subsequently form hyphae (No DOX, dark lines). (C) Histopathological examination of kidneys retrieved from infected mice confirmed that we were able to manipulate C. albicans/fungal morphology in vivo as well as in vitro. Cartoons represent the in vitro morphology prior to injection and predicted outcomes. Note the evidence of fungal element degradation in the DOX treated mice (pseudohyphal infection samples, third panel from left). Sections are from kidneys retrieved from mice sacrificed on day 3 post-infection (see Fig. 5B).
Figure 4.
Figure 4.
Examination of organ fungal burdens at various time points post-infection. Organs were removed from infected mice, homogenized, and samples plated to obtain fungal burdens (CFU g−1). Median burdens are indicated by a horizontal line. Differences in median burdens between pairs of samples were assessed using the Mann–Whitney Test. Some data points included in the analysis result in a score of zero on a logarithmic scale and are not shown. (A) There were only two statistically significant differences 6 h after infection: between the brain burdens of the Plus DOX and No DOX treated groups following yeast form infection (P = 0.0278) and between the kidney burdens of the Plus DOX treated, pseudohyphal (+/+) and the No DOX treated, yeast form (−/−) infections (P = 0.0196). (B) After 3 days, there were no statistically significant differences between any of the groups (P > 0.5). (C) Fungal burdens were determined in the organs retrieved from doxycycline-treated mice sacrificed at various intervals after infection with yeast form cells. By day 6 post-infection there were much fewer fungal organisms recovered from the tissues than at day 3 and by day 15, no fungal organisms were detectable in the spleen and only very few remained in some of the brain samples. (D) Fungal burdens were examined in the organs retrieved from mice infected with the tet-NRG1 strain maintained in the yeast form (No DOX) and sacrificed after 27 days. Even after this extended period, there are numerous fungal organisms present in the brain and kidney.
Figure 5.
Figure 5.
Immunization assays. Mice were infected with one of the three indicated strains (or saline) and subsequently challenged with a wild-type strain (CAF2-1). Inoculation with the tup1Δ strain (approximately 4.3 × 105 CFU) did not provide protection from the subsequent wild-type strain challenge (approximately 5.2 × 105 CFU), with no statistically significant difference between the survival curves of this strain and the saline control (P = 0.6475). Immunization with the nrg1Δ mutant strain (approximately 4.7 × 105 CFU) did offer some protection and significantly altered the survival kinetics (P = 0.0348). Immunization with our regulatable tet-TUP1 strain (approximately 4.3 × 105 CFU, mice treated with DOX) also caused a significant alteration in the survival times (P = 0.0002) compared to the mock-immunized control when challenged with a wild-type strain (approximately 2.8 × 105 CFU).
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