generation-of-fluorescent-protein-fusions-in-candida-species

Generation of Fluorescent Protein Fusions in <em>Candida</em> Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

University of Minnesota

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 "fine-tunes" the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis.

Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans.

Hyphae of filamentous fungi maintain generally linear growth over long distances. In Candida albicans, hyphae are able to reorient their growth in the direction of certain environmental cues. In previous work, the C. albicans bud-site selection proteins Rsr1 and Bud2 were identified as important for hyphae to maintain linear growth and were necessary for hyphal responses to directional cues in the environment (tropisms). To ask if hyphal directional responses are general functions of all yeast bud-site selection proteins, we studied the role of Rax2, ortholog of the Saccharomyces cerevisiae bud-site selection protein Rax2, in C. albicans hyphal morphogenesis. Rax2-YFP localized to the hyphal cell surface in puncta and at the hyphal tip in a crescent. Strains lacking Rax2 had hyphal morphologies that did not differ from control strains. In non-cued growth conditions, rax2 mutant strains had defects in both yeast (bud) and hyphal (branch) site selection and mutant hyphae exhibited non-linear growth trajectories as compared to control hyphae. In contrast, when encountering a directional environmental cue, hyphae lacking Rax2 retained the ability to reorient growth in response to both topographical (thigmotropism) and electric-field (galvanotropism) stimuli but exhibited a reduced ability to establish hyphal growth in the direction of a cathodal stimulus. In conclusion, these results indicate that C. albicans Rax2 is important for establishing sites of emergence of yeast and hyphal daughters and for maintaining the linearity of hyphal growth. In contrast to Rsr1 and Bud2, Rax2 is not involved in responses that require a reorientation of the direction of already established hyphal growth (tropisms). Thus, it appears that some hyphal directionality responses are separable in that they are mediated by a different set of polarity proteins.

Rax2 is important for directional establishment of growth sites, but not for reorientation of growth axes, during Candida albicans hyphal morphogenesis.

Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca(2+) influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca(2+) influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca(2+). Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca(2+). Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca(2+). The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca(2+) influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca(2+)-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance.

Cdc42 GTPase dynamics control directional growth responses.

Human milk oligosaccharides (HMOs) are a highly abundant, diverse group of unique glycans that are postulated to promote the development of a protective bacterial microbiota in the intestine and prevent adhesive and invasive interactions of pathogenic bacteria with mucosal epithelia. Candida albicans, a prevalent fungal colonizer of the neonatal gut, causes the majority of fungal disease in premature infants and is highly associated with life-threatening intestinal disorders.

Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells.

Candida parapsilosis is a common cause of invasive candidiasis, especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans, relatively few have been developed for C. parapsilosis. In this study, we show that plasmids harbouring green, yellow and mCherry fluorescent protein sequences, previously developed for expression in C. albicans, can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis, which are typically prototrophic, because the plasmids include NAT1, a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers who require the ability to visualize C. parapsilosis directly, e.g. in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models.

Article	Year
Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans. Eukaryotic cell\| PubMed ID: 23223038	2013
Rax2 is important for directional establishment of growth sites, but not for reorientation of growth axes, during Candida albicans hyphal morphogenesis. Fungal genetics and biology : FG & B\| PubMed ID: 23608319	2013
Cdc42 GTPase dynamics control directional growth responses. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24385582	2014
Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells. The Journal of nutrition\| PubMed ID: 26180242	2015
PCR-mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis. Yeast (Chichester, England)\| PubMed ID: 26551241	2016

Sara Gonia

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