, Vladislav Raclavsky aAims. Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. Methods. Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. Results. We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole.Conclusions. Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.

Growth patterns of Cryptococcus neoformans submerged culture in different culture volumes, intensity of agitation and types of sealing were evaluated to better understand the physiological role of hypoxia response in this yeast. When low intensity agitation was set at high culture volumes and air exchange between the cultivation vessel and external environment was not abolished completely, the cells proliferated slowly but steadily. On the other hand, when the intensity of agitation was high but the vessel was withheld from fresh air supply, the cells first proliferated rapidly, then arrested completely and finally died. Therefore, the central strategy of C. neoformans here seems to lie in its proliferation-rate adjustment to the available oxygen levels and not in its capacity to survive under anoxia. The data support the opinion that the cultures grown under limited aeration (even though not completely withheld from fresh air supply) are much closer to the real cryptococcal life in human tissues than conventional well-aerated exponential cultures.

Background:Cryptococcus neoformans is an obligate aerobic pathogenic yeast causing lung infection typically followed by spread to the central nervous system. During pathogenesis, it relies on well-established virulence factors. This review focuses on the emerging role of cryptococcal adaptation to hypoxia in pathogenesis. Methods and Results:We examined the MedLine database for information on the cryptococcal hypoxia response. While several recent papers describe components of two presumable hypoxia-sensing pathways including description of their target genes, a link of this system to the hypoxic tuning of proliferation is still missing. In addition, an interpretation of this knowledge in respect to the general picture of microbial pathogenesis is lacking.Conclusions: There seems to be a striking parallel between biofilm formation in bacteria, which results in chronic dormant infection with the potential for acute outbreaks, and the dormant state of primary infection followed by secondary outbreaks in C. neoformans. We propose a hypothesis that cryptococcal response to hypoxia might be the driving force for developing a state of dormant infection which is characterized by slowed proliferation and extensive changes in transcriptome and phenotype. This state enables C. neoformans to survive in host and possibly develop life-threatening acute outbreaks later. Hence, conventional well-aerated planktonic culture is not a good in vitro model for studying the pathogenesis of infection and we advocate the development of a more adequate model. Our further conclusion is that the ability of the immune system and antifungal agents to cope with hypoxia-adapted cells is crucial for the successful eradication of cryptococcal infection.

Cryptococcus neoformans was grown in 96-well microtiter plates sealed by foil which is less than 0.01 % permeable to oxygen. On day 14 of the cultivation, we observed peculiar clusters of small droplike daughter cells arranged around < or = 4 % of mother cells. The fact that most of the other cells had died indicates that few cells had been able to survive hypoxic conditions and escape the cell-cycle arrest. However, their daughters were unable to separate from them and to continue their proliferation under such conditions.

We have reported that the cell cycle behavior of the pathogenic yeast Cryptococcus neoformans (C. neoformans) is different from the cell cycle control exhibited by the model yeast Saccharomyces cerevisiae, and also have reported the molecular characterization and physiological roles of the two main eukaryotic cell cycle genes, C. neoformans cyclin dependent kinase 1 (CnCdk1) and cyclin homologues. We also identified components of the G2‐M control machinery, Wee1 kinase and Cdc25 phosphatase homologues, in C. neoformans. Wee1 and Cdc25 are involved in the inhibition and activation, respectively, of the Cdk1‐cyclin complex at the mitotic entry checkpoint in eukaryotes.On the other hand, in the obligate aerobic pathogenic yeast C. neoformans, limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2‐arrest. The ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. We have identified and characterized the gene that is necessary for the proliferation slowdown and G2‐arrest caused by limited aeration. Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The gene coding for a zinc‐finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1‐dependent (SP1‐like) transcription factors. We have confirmed the role of the cryptococcal homologue of CRZ1/SP1‐like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.Support or Funding InformationThis work was partially supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and Cooperative Research Program of Medical Mycology Research Center, Chiba University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.