bioRxiv preprint doi: https://doi.org/10.1101/435842; this version posted October 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dylag, Colon-Reyes, and Kozubowski 1 2 3 4 Fetal bovine serum-triggered Titan cell formation and growth inhibition are unique 5 to the Cryptococcus species complex 6 7 Mariusz Dylaga,b, Rodney Colon-Reyesa, and Lukasz Kozubowskia* 8 aDepartment of Genetics and Biochemistry, Clemson University, Clemson, SC 29631, US, 9 bInstitute of Genetics and Microbiology, University of Wroclaw, Wroclaw, Poland 10 11 Address for correspondence to 12 Lukasz Kozubowski, [email protected] 13 14 15 16 17 1 bioRxiv preprint doi: https://doi.org/10.1101/435842; this version posted October 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dylag, Colon-Reyes, and Kozubowski 18 ABSTRACT 19 In the genus Cryptococcus, C. neoformans and C. gattii stand out by the number of virulence 20 factors that allowed those fungi to achieve evolutionary success as pathogens. Among the factors 21 contributing to cryptococcosis is a peculiar morphological transition to form giant (Titan) cells. 22 Formation of Titans has been described in vitro. However, it remains unclear whether non-C. 23 neoformans/non-C. gattii species are capable of titanisation. Based on a survey of several 24 basidiomycetous yeasts, we propose that titanisation is unique to C. neoformans and C. gattii. In 25 addition, we find that under in vitro conditions that induce titanisation, fetal bovine serum (FBS) 26 possesses activity that inhibits growth of C. gattii and kills C. neoformans if incubation is 27 conducted in the absence of 5% CO2. Moreover, lowering of pH or addition of an antioxidant, 28 rescues growth in the presence of FBS and allows titanisation even in the absence of 5% CO2. 29 Strikingly, acapsular mutants, cap10Δ and cap59Δ, show complete or partial reduction of 30 titanisation, respectively and a partial resistance to growth inhibition imposed by the FBS. Our 31 data implicate titanisation as a unique feature of C. neoformans and C. gattii and provide novel 32 insights about the nature of this unusual morphological transition. 33 34 35 36 37 38 2 bioRxiv preprint doi: https://doi.org/10.1101/435842; this version posted October 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dylag, Colon-Reyes, and Kozubowski 39 INTRODUCTION 40 Basidiomycetous yeasts are an ecologically heterogeneous group of fungi that includes both 41 saprophytic as well as pathogenic species. A significant fraction of the latter group comprises of 42 human and animal pathogens, including genera of Cryptococcus, Malassezia, Rhodotorula and 43 Trichosporon 1. In particular, two species belonging to the Cryptococcus genus (formerly 44 Filobasidiella), C. neoformans and C. gattii are the etiological agents of fatal systemic mycoses. 45 In recent years, nearly one million cases of cryptococcal meningitis occur annually resulting in 46 over 600,000 deaths globally 2. Moreover, cryptococcosis is responsible for 15-17% AIDS- 47 related deaths on a global scale 3,4. While C. neoformans can cause mainly opportunistic 48 infections in immunocompromised patients, C. gattii is capable of infecting also 49 immunocompetent individuals 5-8. Moreover, it is well established that among C. neoformans, 50 serotype A (varietas grubii) is responsible for the majority of cryptococcal infections, while 51 serotype D (varietas neoformans) is less common 7. Among Cryptococcus species other than C. 52 neoformans and C. gattii, the following species have been described as causing occasional 53 infections in humans: C. laurentii 9-11, C. albidus 9,12,13, C. curvatus 14,15, C. uniguttulatus 16, and 54 C. adeliensis 17. Such casuistic infections, reviewed in literature most recently by Smith et al. 18 55 can be also systemic in case of strains able to grow at 37°C. What makes those selected 56 Cryptococcus species capable of infecting humans is an important question, the answer to which 57 remains incomplete. Among the best-described cryptococcal characteristics necessary for 58 pathogenicity are the ability to proliferate at 37°C, melanisation, formation of capsule, and the 59 capability of hydrolyzing urea 8,19. Most of the above-mentioned “virulence factors” can be 60 observed together only in case of C. neoformans and C. gattii, what could potentially explain the 61 basis of their evolutionary success as pathogens. 3 bioRxiv preprint doi: https://doi.org/10.1101/435842; this version posted October 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dylag, Colon-Reyes, and Kozubowski 62 Dimorphism is one of the common features of human fungal pathogens 20,21. A well- 63 documented example of a dimorphic switching critical for pathogenicity is yeast to hypha 64 transition characteristic for dimorphic fungi (clustered within five genera) and Candida albicans 65 20,21. Morphological transition of C. neoformans to form enlarged cells termed Titan cells is a 66 particularly striking manifestation of a perfect adaptation to evade the mammalian immune 67 system and enhance dissemination in the host 22. Titan cells are resistant to phagocytosis due to 68 over ten-time enlargement of the cell body 22-25. Moreover, it has been demonstrated that Titan 69 cells and aneuploid daughter cells of Titans show increased resistance to many physico-chemical 70 factors and some drugs commonly used in the therapy of cryptococcosis 26. However, titanisation 71 may not be required for pathogenicity as some clinical isolates of C. neoformans are presumably 72 not capable of undergoing this morphological transition 27-29. Therefore, it remains unclear to 73 what extend titanisation is important for cryptococcosis and whether this striking characteristic is 74 unique to pathogenic Cryptococcus species, especially since titanisation has never been studied in 75 species other than C. neoformans and C. gattii. 76 Recent investigations led to considerable advances in our understanding of the nature of 77 titanisation, by uncovering external stimuli that are sufficient and genes that are essential for this 78 morphological transition 27-29. Two well-documented pathways involved in titanisation are the 79 cAMP-mediated signaling (dependent on Gpr4/Gpr5 receptors, adenylyl cyclase Cac1, Pka1 80 kinase, and the transcription factor Rim101) and the mating pathway 30-32. Until recently the 81 progress in identifying more regulators has been hampered by the lack of a suitable in vitro 82 system. However, recently published work from several laboratories delivered new opportunities 83 towards thorough understanding of titanisation by developing in vitro conditions capable of 84 stimulating this morphological change 27-29. This breakthrough research led to identification of 85 novel positive (Gat201, Ada2, Cap59, Cap60, Ric8, Sgf29, Lmp1) and negative (Usv101, Pkr1, 4 bioRxiv preprint doi: https://doi.org/10.1101/435842; this version posted October 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dylag, Colon-Reyes, and Kozubowski 86 Tsp2) regulators that were important for titanisation under specific conditions 27,29. These studies 87 also led to an overarching conclusion that titanisation can be stimulated by a variety of external 88 signals, including CO2, hypoxia, exposure to serum (specifically two serum components, 89 phospholipids and bacterial peptidoglycan), and quorum sensing 27-29. Importantly, these novel 90 protocols to induce titanisation in vitro have provided an opportunity to test to what extent is the 91 ability to form Titans conserved in other Cryptococcus species. 92 Based on a survey of 23 strains that represent 9 non-neoformans species, we postulate that 93 C. neoformans and C. gattii are unique among other basidiomycetous yeasts with regard to the 94 ability to form bona fide Titan cells in the presence of fetal bovine serum. Our work provides 95 novel insights with respect to external factors that are necessary, sufficient, or inhibitory towards 96 titanisation. We uncover that fetal bovine serum contains both inhibitory and stimulatory factors 97 and a balance between these factors determines the ability to form Titan cells. The inhibitory 98 factor affects growth through increase of ROS. In contrast the stimulatory factor triggers 99 titanisation likely through cAMP pathway, consistent with previous findings. Furthermore, 100 exposure to 5% CO2 counteracts the inhibitory effect of serum and further stimulates Titan cell 101 formation. Strikingly, C. gattii and even more significantly C. neoformans var. grubii are 102 particularly sensitive towards the inhibitory effect of serum as compared to other non-neoformans 103 Cryptococcus species. We also demonstrate that deletion of genes involved in capsule formation, 104 CAP10 and CAP59, have differential effect on titanisation; CAP10 is essential and CAP59 105 dispensable for this morphogenetic transition. Our data suggest that C. neoformans and C. gattii 106 have evolved as unique species capable of undergoing titanisation under human host conditions. 107 108 109 5 bioRxiv preprint doi: https://doi.org/10.1101/435842; this version posted October 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dylag, Colon-Reyes, and Kozubowski 110 RESULTS 111 C. neoformans and C. gattii are unique in their ability to undergo titanisation in the 112 presence of fetal bovine serum (FBS) 113 Three in vitro protocols to induce formation of bona fide Titan cells in C. neoformans have been 114 published recently 27-29.