P381 Kinetics of Titan cells generation and transcriptome modifications comparing three in vitro protocols
Authors: Leclere Aude Sturny , Lemee Laure , Cokelaer Thomas , Alanio Alexandre 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
To face and escape his environment and the host immune response, Candida neoformans is able to change his morphology (Titan cells) and his metabolism (dormancy, quiescence). Titan cells (TC) have been observed in lungs and brains in experimental mouse models of cryptococcosis and in patients. In 2018, three independent teams published three protocols allowing the reproducible generation of TC in vitro [Hommel B et al., (AA); Trevijano-Contador N, et al., (OZ), and Dambuza IM et al., (EB), in PLoS Pathogens 2018]. TC generated in these protocols exhibited the same features as the TC described in vivo. Our objective was to compare and describe the three protocols in parallel to highlight common and different features that can impact further study using those specific protocols.

Methods
A total of 22 h before starting the kinetics of the TC generation, the three protocols requested a pre-culture at 30°C in three different liquid media. The medium for TC production for the three labs was also different but a common factor is the addition of fetal calf serum (FBS) for OZ and EB. This kinetics was evaluated for size and quantity (%) of TC produced over a 72 h period (H0, H18, H24, H48, H72) at 30°C under shaking for AA while OZ and EB protocols incubate the cells at 37°C and 5% of CO2, while the whole transcriptome was analyzed at H0, H3, H7, and H18 in triplicates.

Results
OZ generated the highest percentage of TC, 63.1% and 58.2% at H18 and H24, and decreased drastically down to 6.7% at H48. EB reached a high percentage of TC at H24 for 46.7% and dropdown &lt;10% until the end of the kinetics. AA did not reach a quantity of TC as high as the two other protocols but it remained constant over a period of H72 (Table 1). RNA sequencing preliminary analysis showed some differences in genes expressed at the different time points analyzed. The PCA analysis revealed that the triplicates of each protocol for the 4-time points analyzed are closed to each other, related to the good quality of our experiments. The differential gene expression (DGE) showed significant (P &lt;.01 and Log2 fold change &gt;1) differences at H0 which highlights the impact of the preculture on the TC process. The highest numbers of DGE are observed between H0 and H7 for the three protocols, where about two 450 DGE, two 000 DGE, and two 300 AA, OZ, and EB, respectively. After analysis of the PCA plot during the kinetics, EB and OZ are grouped while AA is not. That could be explained by the presence of FBS in OZ and EB protocols.

Conclusion
By running the three protocols in parallel, we showed here that the kinetics of TC generation differed between each other with a significant variation of the transcriptome. This is an important finding that paves the way to compare more deeply the transcriptome of C. neoformans during TC generation with the final goal is to identify the genes associated with TC generation.