Ras1-mediated Morphogenesis in the Human Fungal Pathogen Cryptococcus Neoformans

<p><italic>Cryptococcus neoformans</italic> pathogenesis results from the proliferation of yeast-phase fungal cells within the human host. The Ras1 signal transduction cascade is a major regulator of <italic>C. neoformans</italic> yeast and hyphal-phase morphogenesis, thermotolerance, and pathogenesis. Previous work identified the conserved Rho-GTPases Cdc42 and Rac1 as potential downstream targets of Ras1. In this work, we identify the duplicate Cdc42 and Rac paralogs, Cdc42 and Cdc420, and Rac1 and Rac2, as major effectors of Ras1-mediated thermotolerance and polarized growth, respectively. Using genetic and molecular biology techniques, including mutant analyses and over-expression studies, we determine the separate and overlapping roles of the four Rho-GTPases in Ras1-mediated morphogenesis. The Cdc42 paralogs are non-essential but are required for thermotolerance and pathogenesis. Ras1 acts through the Cdc42 paralogs to regulate cytokinesis via the organization of septin proteins. The major paralog, Cdc42, and the minor paralog, Cdc420, exhibit functional differences that are primarily dictated by transcriptional regulation. Additionally, CDC42 transcription is induced by exposure to temperature stress conditions. In contrast, Ras1 acts through the equivalently transcribed RAC paralogs to regulate polarized growth during both yeast and hyphal-phase morphogenesis. Rac1 and Rac2 are individually dispensable and appear to be functionally redundant but are synthetically required for yeast phase growth and spore development. The sub-cellular localization of the Rac paralogs is dependent on both Ras1 and post-translational modification by prenyl transferases. The identification and characterization of the conserved elements of the Ras1 signal transduction cascade presented here constitute an important contribution towards the design of anti-fungal agents that are based on existing Ras-pathway inhibitors.</p>Dissertatio

Ras1 Acts through Duplicated Cdc42 and Rac Proteins to Regulate Morphogenesis and Pathogenesis in the Human Fungal Pathogen <i>Cryptococcus neoformans</i>

<div><p>Proliferation and morphogenesis in eukaryotic cells depend on the concerted activity of Rho-type GTPases, including Ras, Cdc42, and Rac. The sexually dimorphic fungus <i>Cryptococcus neoformans</i>, which encodes paralogous, non-essential copies of all three, provides a unique model in which to examine the interactions of these conserved proteins. Previously, we demonstrated that <i>RAS1</i> mediates <i>C. neoformans</i> virulence by acting as a central regulator of both thermotolerance and mating. We report here that <i>ras1Δ</i> mutants accumulate defects in polarized growth, cytokinesis, and cell cycle progression. We demonstrate that the <i>ras1Δ</i> defects in thermotolerance and mating can be largely explained by the compromised activity of four downstream Rho-GTPases: the Cdc42 paralogs, Cdc42 and Cdc420; and the Rac paralogs, Rac1 and Rac2. Further, we demonstrate that the separate GTPase classes play distinct Ras-dependent roles in <i>C. neoformans</i> morphogenesis and pathogenesis. Cdc42 paralogs primarily control septin localization and cytokinesis, while Rac paralogs play a primary role in polarized cell growth. Together, these duplicate, related signaling proteins provide a robust system to allow microbial proliferation in the presence of host-derived cell stresses.</p></div

Ras1 acts through Cdc42 and Rac paralogs to regulate polarized growth and morphogenesis.

<p>GTP-bound Ras1 preferentially interacts with the Rho-GEF Cdc24 and mediates the activation of Rac1/Rac2 and Cdc42/Cdc420. The Rac paralogs primarily regulate polarized growth and organize the localization of ROS during filamentous growth, possibly through interaction with as yet unidentified Nox proteins. Cdc42 paralogs are primarily involved in cytokinesis and bud morphogenesis via the organization of the septin proteins. Additionally, Rac and Cdc42 paralogs physically interact with both Pak1 and Ste20.</p

Analysis of bud morphology.

<p>Log-phase cultures of the indicated cell types were incubated at 37°C for 24 hours or 4 hours in liquid medium, 150 RPM. DIC images of the cultures were analyzed for mother and daughter cell morphology. For 4 hour cultures, a sufficient number of cells (n_t) was examined to identify comparable numbers of budding cells (n_b). Abnormal buds included those that were non-round or had wide bud necks, and mother cells with multiple buds or buds that were abnormally large (>75% of the size of the mother cell).</p

Septin localization in <i>C. neoformans</i> cells at 30 and 37°C after 4 hours.

<p>Localization of the Cdc11-mCherry fusion construct in various cell backgrounds incubated at the indicated conditions for 4 hours. A) In wild type cells, septin localization undergoes predictable changes in organization from a patch in the early emerging bud (*) to the hourglass arrangement on either side of the neck (←) during bud development, to the double ring structure during cytokinesis ( = ). B) Localization is lost in the majority of <i>ras1Δ</i> cells at 37°C, although aberrant localization can be observed in a minority of cells (2.7%). C) Wild type, <i>ras1Δ</i>, <i>ras1Δ+RAC2</i>, <i>ras1Δ+CDC42</i>, <i>ras1Δ+CDC420</i>, and <i>ras1Δ+RAC1</i> strains were spotted in 5 fold dilutions starting with 10⁶ cells/ml. Plates were incubated for 48 hours at 30°C or 37°C and then photographed. D) The loss of the septin <i>CDC3</i> results in the accumulation of cytokinesis defects at both 30 and 37°C, with cells forming chains of un-separated daughters. The over-expression of <i>RAC1</i> restores polarized growth and bud development to the <i>ras1Δ</i> mutant, but is insufficient to restore cytokinesis. Like <i>cdc3Δ</i> cells, <i>ras1Δ+RAC1</i> cells accumulate defects in cytokinesis, with daughter cells failing to separate. E) The over-expression of <i>CDC42</i> paralogs restores Cdc11-mCherry localization at 37°C. F) The over-expression of <i>RAC2</i> fails to restore Cdc11-mCherry localization at 37°C.</p

Mating in <i>ras1Δ+RhoGTPase</i> strains.

<p>Wild type (A), <i>ras1Δ</i> (B), <i>ras1Δ+RAC1</i> (C), <i>ras1Δ+RAC2</i> (D), <i>ras1Δ+CDC42</i> (E), and <i>ras1Δ+CDC420</i> (F) mating type α cells were co-incubated with wild type mating type <b>a</b> cells on MS mating medium for 4 days. Wild type mating reactions results in the formation of hyphal filaments that extend into the medium, while <i>ras1Δ</i> cells are sterile. The over-expression of <i>RAC</i> paralogs restores filamentation. Arrows indicate abnormal clamp connections (←), and stars (*) indicate fused clamp connections. The over-expression of <i>CDC42</i> paralogs fails to restore filamentation to the <i>ras1Δ</i> strain. Scale bar = 50 µm.</p

Strains in this work.

<p>Strains in this work.</p

Clamp cell morphogenesis during <i>C. neoformans</i> hyphal development.

<p>(A) Schematic of clamp cell fusion and nuclear dynamics. (B) Clamp cell morphogenesis depends on the fusion of clamp and peg cells across hyphal compartments. (C, D) The over-expression of <i>RAC1</i> (C) or <i>RAC2</i> (D) is insufficient to restore clamp cell morphogenesis in the <i>ras1Δ</i> background compared to wild type clamp cells (top panels). (E) During <i>RAS1α</i>×<i>RAS1</i><b><i>a</i></b> mating, hypha with fused clamp cells are dikaryotic. (F) In mating filaments produced by the <i>ras1Δ+RAC2α×RAS1</i><b><i>a</i></b> cross, nuclei were observed trapped in the unfused clamp cell.</p

Yeast phase morphology of <i>C. neoformans</i> cells at 30 and 37°C after 4 hours.

<p>Wild type (A), <i>ras1Δ</i> (B), <i>ras1Δ+CDC42</i> (C), and <i>ras1Δ+RAC2</i> (D) cells were incubated at the indicated conditions for 4 hours. A) Wild type cells proliferate by budding at both 30 and 37°C; however, at 37°C wild type cells increase in size overall compared to 30°C. B) Mutant <i>ras1Δ</i> cells are larger than wild type cells at both 30 and 37°C. At 37°C, a population of <i>ras1Δ</i> budding cells (57.5% vs. 66.2% in wild type) was observed, 42.9% of which were abnormal in morphology (vs. 2.8% in wild type cells). Abnormal morphology included mother cells with multiple buds (*) or abnormally large buds (8) (>75% as large as mother cells), as well as buds with wide necks or abnormal morphology (←). C) The over-expression of <i>CDC42</i> restores wild type morphology to the majority of <i>ras1Δ</i> buds at 37°C (72.5% budded, 11.2% abnormal), but does not restore cell size. D) The over-expression of <i>RAC2</i> restores cell size to <i>ras1Δ</i> cells at 30°C and 37°C, but does not support cell separation. Scale bar = 10 µm for all panels.</p