Exploring the mechanism of cytokinesis failure in Cryptococcus neoformans treated with an antifungal drug, fluconazole
Diana Fang and Lukasz Kozubowski
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634 USA
Abstract
Cryptococcus neoformans is a pathogenic yeast that causes lethal cryptoccocal meningitis in immunocompromised patients. One of the challenges in treating cryptococcosis is in its ability to develop resistance to an antifungal drug, fluconazole, which is used to treat it. In previous studies, we found that fluconazole leads to the failure of cytokinesis and/or final cell separation during mitosis, which may contribute to fluconazole resistance. To investigate which part of cytokinesis has failed in C. neoformans treated with fluconazole, we monitored the dynamics of the key cytokinesis component, the actomyosin ring (AMR). Our data show heterogeneous responses to fluconazole in terms of timing and rate of constriction of the AMR. After the constriction of the AMR, a septum was usually formed between the mother and daughter cells. Therefore, these findings suggest that even in the presence of fluconazole, the AMR does constrict and a septum is formed. However, a final degradation of the septum between mother and daughter may not occur, resulting in the lack of complete separation between the cells.
Background
- Fungal pathogens such as Cryptococcus neoformans pose a great threat to the lives of those who are immunocompromised (Brown, et al., 2012). C. neoformans causes pneumonia and meningitis that is responsible for approximately 620,000 deaths per year (Park et al., 2009).
- The deaths that occur from cryptococcosis results from the ability of C. neoformans to develop resistance to anti-fungal drug used to treat it, fluconazole (FLC) (Sionov et al., 2012). In previous studies, formation of trimeric cells, where three cells do not separate, appeared after 5-6 hours of treatment with FLC. The multibudded phenotype suggests that at least one of the daughter cells did not complete cytokinesis. This phenomenon may contribute to the resistance of FLC.
- Cytokinesis is the process by which the cellular contents of the mother cell get partitioned into the daughter cell during cell division. In fungal cells, cytokinesis involves the contraction of the actomyosin ring (AMR), coupled with the formation of the septum and a final degradation of the septum (Meitinger & Palani, 2016). In this study, we hypothesized that the failure in cytokinesis at the level of AMR contributes to the resistance to FLC observed in C. neoformans.
Materials and Methods
- Strains, Media, Growth Conditions. A derivative of the C. neoformans var. grubii strain, LC4, was used in this study. LC4 was grown in yeast-peptone-dextrose (YPD) media at 24 ˚C.
- Treatment with Fluconazole (FLC). LC4 was grown overnight. A density of 2.55 x 106 cells/mL was passed back and 24 µg/mL FLC added. Yeast cells were incubated for 6 to 7 hours in FLC prior to each experiment.
- Microscopy. Six hundred microliters of cell suspension containing yeast-nitrogen-base (YNB) liquid media with ~1.50 x 106 cells/mL in 24 µg/mL FLC was placed in a chamber of a borosilicate 4-chamber-slide (Bio-Tek, Winooski, VT). Large budded yeast cells were targeted and imaged every 15 – 20 minutes. The bright field and fluorescence images were captured with a Zeiss Axiovert 200 inverted microscope (Carl Zeiss, Thornwood, NY) and interfaced with AxioVision Rel 4.8 software (Carl Zeiss, Thornwood, NY). Images were processed in Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) and organized in CorelDRAW Suite X4 (Corel Corporation, Ottawa, ON, Canada).
Budding Yeast
Normal FLC Treated
Results
mCherry-Myo1 localized at the bud neck of both daughters within a trimera. We were able to image the constriction of the AMR of the first daughter cell of the trimera and it took approximately 35 minutes, and has formed at 15 minutes (Figure 1).The constriction of the AMR in the second daughter cell of the trimera took up to 75 minutes (Figure 2). Towards the end of AMR constriction (95 min time point in Figure 1), a septum formed. The rate of the constriction of AMR in the second daughter differed from cell to cell and ranged between 10 and 75 minutes (Figure 3).
Figure 1 Constriction of AMR in first daughter takes place in approximately 35 minutes.
Figure 2 Constriction of AMR in second daughter cell takes place in approximately 70 minutes.
Figure 3 Time taken for AMR construction in the second daughters (n = 6). Half of the trimeras observed took between 16-30 minutes for the AMR to constrict.
To view an animated version of Figure 2, please Click here
Objectives
- To elucidate the dynamics of AMR during cytokinesis in yeast cells treated with FLC.
- To determine if cytokinesis had failed in either of the daughter cells, which are parts of the multibudded trimera.
Conclusion
Our data show that the AMR does constrict in the presence of fluconazole but the rate at which constriction occurs varies between cells. Constriction occurs following the formation of both daughters. A septum is also formed, but a final degradation of the septum between mother and daughter cells may not occur, resulting in the lack of separation between the mother and daughter.
Future studies include monitoring the dynamics of other proteins involved in cytokinesis in C. neoformans such as septins to see if the failure in septin localization at the bud-neck would contribute to the failure of separation between mother and daughter yeast cells, and thus leading to the resistance of FLC.
Acknowledgements
We thank Logan Crowe for generating the LC4 strain.
This study was funded by the 1R15 AI119801-01 grant from the NIH.
References
Brown, G. D., Denning, D. W., & Levitz, S. M. (2012). Tackling Human Fungal Infections. Science, 336(6082), 647–647. http://doi.org/10.1126/science.1222236
Meitinger, F., & Palani, S. (2016). Actomyosin Ring driven Cytokinesis in Budding Yeast. Seminars in Cell & Developmental Biology. http://doi.org/10.1016/j.semcdb.2016.01.043
Park, B. J., Wannemuehler, K. A., Marston, B. J., Govender, N., Pappas, P. G., & Chiller, T. M. (2009). Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS, 23(4), 525–530. http://doi.org/10.1097/QAD.0b013e328322ffac
Sionov, E., Chang, Y. C., Garraffo, H. M., Dolan, M. A., Ghannoum, M. A., & Kwon-Chung, K. J. (2012). Identification of a Cryptococcus neoformans cytochrome P450 lanosterol 14α-demethylase (Erg11) residue critical for differential susceptibility between fluconazole/voriconazole and itraconazole/posaconazole. Antimicrobial Agents and Chemotherapy, 56(3), 1162–9. http://doi.org/10.1128/AAC.05502-11
Abbreviations
AMR: actomyosin ring; FLC: fluconazole; YNB: yeast-nitrogen-base; YPD: yeast-peptone-dextrose