A Novel Resistance Pathway for Calcineurin Inhibitors in the Human

bioRxiv preprint doi: https://doi.org/10.1101/834143; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 1 A novel resistance pathway for calcineurin inhibitors in the human 2 pathogenic Mucorales Mucor circinelloides 3 4 Sandeep Vellanki1, R. Blake Billmyre2, 3, Alejandra Lorenzen1, Micaela Campbell1, 5 Broderick Turner1, Eun Young Huh1, Joseph Heitman3, and Soo Chan Lee1# 6 7 1South Texas Center for Emerging Infectious Diseases (STCEID), Department of 8 Biology, The University of Texas at San Antonio, San Antonio, TX. 9 2Current address: Stowers Institute for Medical Research, Kansas City, MO. 10 3Department of Molecular Genetics and Microbiology, Duke University Medical Center, 11 Durham, NC. 12 13 #Corresponding author: 14 Soo Chan Lee, Ph.D. 15 1 UTSA Circle, 16 South Texas Center for Emerging Infectious Diseases (STCEID), 17 Department of Biology, 18 University of Texas at San Antonio, San Antonio, TX 78249, USA 19 Email: [email protected] 20 Phone: (210) 458-5398 21 22 (Word count: Abstract: 446; Text: 4821) 23 Running title – Resistance to calcineurin inhibitors in Mucorales 1 bioRxiv preprint doi: https://doi.org/10.1101/834143; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 24 Abstract 25 Mucormycosis is an emerging lethal fungal infection in immunocompromised 26 patients. Mucor circinelloides is a causal agent of mucormycosis and serves as a model 27 system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor 28 in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin 29 regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We 30 analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal 31 growth in the presence of calcineurin inhibitors or in the yeast-locked cnbR mutant 32 background without carrying any mutations in known calcineurin components. We found 33 that a majority of the mutants had altered sequence in a gene, named here bycA 34 (bypass of calcineurin A). bycA encodes an amino acid permease. We verified that both 35 bycA, and the bycA cnbR double mutant are resistant to the calcineurin inhibitor 36 FK506, thereby demonstrating a novel resistance mechanism against calcineurin 37 inhibitors. We also found that the expression of bycA was significantly higher in the wild 38 type strain treated with FK506 and in the cnbR mutants, but significantly lower in the 39 wild type without FK506. These findings suggest that bycA is a negative regulator of 40 hyphal growth and/or a positive regulator of yeast growth in Mucor and calcineurin 41 suppresses the bycA gene at the mRNA level to promote hyphal growth. BycA is 42 involved in the Mucor hyphal-yeast transition as our data demonstrates a positive 43 correlation between bycA expression, protein kinase A activity, and Mucor yeast-growth. 44 Also calcineurin, independent of its role in morphogenesis, contributes to virulence traits 45 including phagosome maturation blockade, host cell damages, and pro-angiogenic 46 growth factor induction during interactions with hosts. 2 bioRxiv preprint doi: https://doi.org/10.1101/834143; this version posted December 19, 2019. 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. 47 Importance 48 Mucor is intrinsically resistant to most known antifungals, which makes 49 mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase 50 widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, 51 growth shifts to a less-virulent yeast growth form which makes calcineurin an attractive 52 target for development of new antifungal drugs. Previously we identified two distinct 53 mechanisms through which Mucor can become resistant to calcineurin inhibitors 54 involving Mendelian mutations in the gene for FKBP12, calcineurin A or B subunits and 55 epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism 56 where loss of function mutations in the amino acid permease encoding the bycA gene 57 contribute to resistance against calcineurin inhibitors. When calcineurin activity is 58 absent, BycA can activate PKA to promote yeast growth via a cAMP-independent 59 pathway. Our data also shows that calcineurin activity, primarily contributes to host - 60 pathogen interactions in the pathogenesis of Mucor. 61 62 63 Introduction 64 Mucormycosis is a severe life-threatening infection caused by fungi belonging to 65 the order Mucorales (1). People with weakened immune systems due to diabetes 66 mellitus, neutropenia, or hematological and solid organ transplantation are at the 67 highest risk of acquiring this infection (2, 3). It is the third most common invasive fungal 68 infection in hematological and allogeneic stem transplantation patients following 69 candidiasis and aspergillosis (1, 4). Over the past decades, there has been a global and 3 bioRxiv preprint doi: https://doi.org/10.1101/834143; this version posted December 19, 2019. 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. 70 ongoing rise in the incidence of mucormycosis, primarily due to the increasing number 71 of diabetic patients and increased use of immunosuppressive drugs (5-10). 72 Mucormycosis is also on the rise in immunocompetent individuals (11-14). Mucorales 73 grow as molds in the environment and produce sporangiospores that can enter the host 74 via inhalation resulting in pulmonary infection, through the skin due to trauma resulting 75 in cutaneous infections, or through the nasal passages resulting in rhinocerebral 76 infections (15-17). The spores can disseminate within the host, resulting in 95-100% 77 mortality even with antifungal drug treatment (18). Mucorales are intrinsically resistant to 78 most antifungals, which makes it very difficult to treat, and surgery is often required 79 (19). 80 Calcineurin is a calcium-calmodulin-dependent phosphatase conserved widely 81 across eukaryotes including pathogenic fungi (20, 21). Calcineurin is a heterodimer 82 consisting of a catalytic and a regulatory subunit, and both subunits are required for 83 calcineurin function. The role of calcineurin varies depending on the fungal species, for 84 example in Cryptococcus neoformans and Cryptococcus gattii calcineurin is required for 85 growth at high temperature (37°C) and at alkaline pH (22-24), while in Candida spp. 86 calcineurin contributes to azole tolerance and is required for survival in serum, among 87 other functions (20, 25). In Aspergillus fumigatus, calcineurin mutants exhibit delayed 88 germination, hyphal growth with irregular branching, and abnormal septum (26). We 89 have previously shown that in Mucor spp. calcineurin regulates dimorphism, in which 90 the calcineurin inhibitor FK506 (tacrolimus) enforces Mucor to grow only as yeast (27). 91 Hyphal morphology is the predominant growth mode for Mucor spp., however by 92 modulating respiratory conditions Mucor can be forced to grow as yeast as well (28). 4 bioRxiv preprint doi: https://doi.org/10.1101/834143; this version posted December 19, 2019. 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. 93 While aerobic conditions promote hyphal growth, low oxygen and high carbon dioxide 94 conditions enforce yeast growth (29-32). Targeting components involved in 95 mitochondrial or lipid metabolism can also promote yeast growth, even under aerobic 96 conditions (33-36). In addition, previous studies have shown that the addition of cyclic 97 AMP to Mucor in culture results in activation of cAMP-dependent kinase Protein Kinase 98 A and promotes yeast growths (37-40). Wolff et al have also shown that during 99 anaerobic yeast growth in Mucor an increased expression of PKA regulatory and 100 catalytic subunits are exhibited than during aerobic hyphal growth (41). Mucor spp have 101 four isoforms of PKA regulatory subunits, and each is differentially expressed 102 depending on the growth conditions (38, 39). Calcineurin is involved in the genetic 103 regulation of Mucor dimorphism as deletion of the gene encoding the regulatory subunit 104 of calcineurin (CnbR) resulted in yeast-locked growth, even under aerobic conditions 105 (27). cnbR mutants are avirulent in a wax moth host model thereby making calcineurin 106 an attractive target for antifungal treatment in mucormycosis (27). Also, mucormycosis 107 incidence is low in patients receiving FK506 as an immunosuppressant (42). cnbR 108 mutants are also more susceptible to antifungal drugs such as Ambisome, micafungin, 109 and posaconazole (43). 110 The cellular receptor for calcineurin is FKBP12, a member of the immunophilin 111 protein family with cis-trans peptide prolyl isomerase activity (44). When FK506 is 112 bound to FKBP12, it inhibits calcineurin phosphatase activity by binding to the interface 113 between the calcineurin catalytic A and regulatory B subunits, thereby preventing 114 substrates access to the active site (45, 46). FKBP12 also binds to rapamycin to inhibit 115 the Tor pathway (47), and mutations in the FKBP12 gene confer resistance to both 5 bioRxiv preprint doi: https://doi.org/10.1101/834143; this version posted December 19, 2019. 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. 116 FK506 and rapamycin. Amino acid substitutions in the calcineurin regulatory B and 117 catalytic A subunit surfaces that interact with the FKBP12-FK506 complex can also 118 result in resistance to FK506 (48, 49). Another immunophilin, cyclophilin A (Cyp), serves 119 as a cellular receptor for the drug cyclosporine A (CsA).

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