Two CDC42 paralogues modulate Cryptococcus neoformans thermotolerance and morphogenesis under host physiological conditions

Abstract

The precise regulation of morphogenesis is a key mechanism by which cells respond to a variety of stresses, including those encountered by microbial pathogens in the host. The polarity protein Cdc42 regulates cellular morphogenesis throughout eukaryotes, and we explore the role of Cdc42 proteins in the host survival of the human fungal pathogen Cryptococcus neoformans. Uniquely, C. neoformans has two functional Cdc42 paralogues, Cdc42 and Cdc420. Here we investigate the contribution of each paralogue to resistance to host stress. In contrast to non-pathogenic model organisms, C. neoformans Cdc42 proteins are not required for viability under non-stress conditions but are required for resistance to high temperature. The paralogues play differential roles in actin and septin organization and act downstream of C. neoformans Ras1 to regulate its morphogenesis sub-pathway, but not its effects on mating. Cdc42, and not Cdc420, is upregulated in response to temperature stress and is required for virulence in a murine model of cryptococcosis. The C. neoformans Cdc42 proteins likely perform complementary functions with other Rho-like GTPases to control cell polarity, septin organization and hyphal transitions that allow survival in the environment and in the host.

Figure 1. C. neoformans species maintain two Cdc42 paralogs

A) Alignment of C. neoformans (Cn) Cdc42 and Cdc420 protein sequences with Cdc42 protein sequences from S. cerevisiae (Sc), S. pombe (Sp), and U. maydis (Um). Key domains are indicated and the C. neoformans Rac1 sequence is provided for comparison. Residue 56 distinguishes Cdc42(F) and Rac(W) proteins by providing specificity with GEFs (Hlubek et al., 2008). B) Phylogenetic tree showing the relatedness of Cdc42 protein sequences from three C. neoformans species, U. maydis C. cinerea, P. marneffei, S. pombe, Candida species, A. fumigatus, S. cerevisiae, and R. oryzae (rooted).

Figure 2. Growth defects of cdc42Δ, cdc420Δ, and cdc42Δ cdc420Δ mutants

Indicated strains were spotted in 5-fold serial dilutions onto solid YPD medium and incubated at 30°, 37°, or 39°C as indicated for 48 hours.

Figure 3. Cdc42 paralogs are implicated in thermotolerance and morphogenesis

A) Wild type, cdc42Δ cdc420Δ, cdc42Δ cdc420Δ, proCDC42:CDC420 and proCDC420:CDC42 strains were inoculated into YPD and grown to mid-log phase. Cultures were split and refreshed with media pre-warmed to 30° or 37°C and allowed to grow under the indicated conditions for 24 hours. Cells were fixed and stained with Calcofluor for imaging. (Bar = 10µm).

Figure 4. CDC42 paralogs play a role in actin re-polarization after exposure to stress

A) Indicated strains were examined in the presence of the actin inhibitor Latrunculin B in a 96 well format. Cells (10⁶ml⁻¹) were incubated in the presence of decreasing concentrations of Latruculin B (100µM to 0µM) for 48 hours (n=3). The concentration of drug that failed to inhibit growth was noted for each strain. B, C) Wild type and cdc42Δ cdc420Δ cells were grown to log phase, and cultures were split and refreshed with pre-warmed media at 30° and 37°C. Cultures were grown at 30° or 37°C. After 120 minutes (B) or 4 hours (C) aliquots were fixed, permeablized, and stained with rhodamine-conjugated phalloidin to reveal actin structures. Budding cells were categorized as polarized or apolar according to previously established criteria (Nichols et al., 2007; Waugh et al., 2002b). (Bar = 10µm)

Figure 5. CDC42 paralogs are differentially expressed

A) Primer pairs specific for CDC420 or CDC42 transcripts were identified and assessed by semi-quantitative RT-PCR in wild type cDNA. B) CDC42 and CDC420 transcripts were detected by real-time PCR in wild type (H99), cdc42Δ, and cdc420Δ strains at 30° and 37°C. Bars represent the fold change in expression between temperature conditions, relative to an internal control (GPD) (n=4). C) Promoter swap constructs were generated and introduced in the double mutant background.

Figure 6. Cdc42 paralogs are involved in septin organization

The localization of a Cdc10-mCherry construct was examined in wild type (A, B) cdc420Δ (C, D) cdc42Δ (E, F) and ste20Δ (G, H) cells grown at 30° or 37°C respectively. Cells were inoculated in to YPD and grown to log phase. Cultures were split, refreshed with pre-warmed media, and allowed to grow for 4 hours. Cells were gently fixed before imaging as described. (Bar = 10µm)

Figure 7. Cdc42 but not Cdc420 is required for clamp cell formation and sporulation

A) Wild type × wild type, cdc42Δ × cdc42Δ, and cdc420Δ × cdc420Δ cells were co-cultured on V8 mating medium and allowed to incubate in the dark at room temperature. The same location on each plate was photographed every 24 hours. B) Basidia heads characteristic of each bilateral cross were identified and spore chains were photographed in situ. C) Slide squashes of mating filaments were prepared as described. Clamps were categorized as normal, abnormal unfused, or abnormal aberrant. 100 clamps were counted for each strain. D) Mating plugs were fixed, permeablized, and stained with Dapi to reveal nuclei.

Figure 8. Cdc42 but not Cdc420 is required for virulence

A/Jcr mice (10 per strain) were inoculated intra-nasally with the indicated strains (5 × 10⁵ cells). Mice were monitored daily for weight loss and neurological symptoms and sacrificed at predetermined clinical endpoints, which correlate with an imminent lethal infection.

Figure 9. Proposed C. neoformans Ras1 pathway for growth and thermotolerance

Ras1 regulates mating, morphogenesis, and thermotolerance via a branched signaling pathway. In this work, we have investigated the contribution of Cdc42 paralogs to morphogenesis and thermotolerance. Based on data presented here, as well as previously published work, we propose a pathway in which Ras1 signals through multiple Rho-GTPases: Cdc42, Cdc420, Rac1, and Rac2; with Cdc42 and Rac1 playing the major roles, and Cdc420, and possibly Rac2, playing minor roles. Rac1 primarily is required for hyphal morphogenesis, and may act through Ste20 and Pak1 to organize the actin cytoskeleton during cytokinesis. Cdc42, and to a lesser extent Cdc420, organize the septins during cytokinesis, and play a role in actin organization during growth under stress conditions. We additionally propose the existence of multiple as yet uncharacterized GEFs, which may provide specificity to Ras1 Rho-GTPase-mediated signaling.