Calcineurin Plays Key Roles in the Dimorphic Transition and Virulence of the Human Pathogenic Zygomycete Mucor circinelloides

Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO2 conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen.


Introduction
Mucor circinelloides is one causal agent of mucormycosis, an uncommon but frequently lethal fungal infection of humans. Several other species belonging to the Mucorales order also cause mucormycosis, including some Rhizopus species, Absidia, Cunninghamella, Rhizomucor, Apophysomyces trapeziformis, and others [1][2][3]. Mucormycosis is an emerging fungal infection often afflicting immunocompromised and vulnerable populations including patients with diabetes, AIDS, hematologic malignancies, or trauma [3][4][5][6][7][8]. Susceptible hosts also include patients with solid organ transplants and those with high serum iron levels [4,8,9]. Mucormycosis is the second most common fungal infection among patients with hematological malignancies and transplants. This infection is associated with high mortality rates [4,8,10]: ,50% for all mucormycosis infections and .90% for disseminated infections [4][5][6]9,11]. Recently, a cluster of mucormycosis cases was reported among the tornado victims in Joplin, MO in the spring of 2011; 13 cases were reported with five fatalities. Based on 28S ribosomal DNA sequence the responsible species has been identified as A. trapeziformis [3]. Despite the increasing incidence of disease, high mortality rate, and unmet clinical needs for therapy, zygomycetes are understudied compared to other pathogenic fungi.
Mucor species are dimorphic fungi and exhibit either hyphal or yeast growth depending upon the conditions (reviewed in [12]). Hyphal growth of Mucor was first thought to originate from species of Saccharomyces by transmutation [13] until Louis Pasteur discovered that Mucor grows as a multi-budded yeast under anaerobic/high CO 2 growth conditions [14]. Later, Bartnicki-Garcia and Nickerson rediscovered the induction of Mucor yeast growth by CO 2 [15][16][17]. Although dimorphic Mucor species vary in their responses to the environment during morphogenic changes, common critical factors that induce yeast growth of Mucor species include oxygen concentration, CO 2 concentration, and carbon source (reviewed in [12]). In addition, several chemicals that inhibit mitochondrial function [including potassium cyanide and antimycin A (which block electron transport) or oligomycin and phenyl alcohol (which inhibit oxidative phosphorylation)] induce yeast growth in Mucor spp., even in aerobic conditions [18][19][20]. Inhibition of the synthesis of cytochrome b and other mitochondrial components by chloramphenicol also results in Mucor yeast growth [21,22]. Thus, respiration, anaerobic conditions, and high CO 2 environments all contribute to the yeasthyphal transition. Cerulenin, a lipid metabolism inhibitor, and cycloleucine, an S-adenosylmethionine (SAM) synthetase inhibitor, both block the yeast to hyphal growth transition under aerobic conditions [23,24]. Notably, adding cyclic AMP (cAMP) to the culture medium induces yeast growth of Mucor spp. (reviewed in [12] and references therein). cAMP activates cAMP-dependent protein kinase A (PKA), implicating a role for protein kinase A in the Mucor dimorphic transition, and this is supported by a series of recent studies, including genetic analyses in which genes encoding PKA regulatory subunits were disrupted by homologous recombination [25][26][27][28][29][30][31]. PKA also plays a key role in various morphogenesis processes including germination, branching, and polarized growth in Mucor [26,[32][33][34][35][36][37] Dimorphism has evolved in multiple lineages in the fungal kingdom, including ascomycetes, basidiomycetes, and zygomycetes [38][39][40]. In many pathogenic fungi, the morphogenic transition is closely related to pathogenicity. A group of so-called ''dimorphic fungi'' grow as molds at lower environmental temperatures; however, in association with the host these fungi undergo a thermal dimorphic switch, resulting in yeast growth as virulent forms at 37uC (reviewed in [41,42]). The dimorphic fungal pathogens include Histoplasma capsulatum, Paracoccidioides brasiliensis, Coccidioides immitis/posadasii, Blastomyces dermatitidis, and Penicillium marneffei, among others. The most prevalent fungal commensal and pathogen, Candida albicans, exhibits yeast and hyphal growth, and the ability of this fungus to grow as both forms is necessary for virulence; yeast-locked or hyphal-locked mutants are both avirulent [39,40,43,44]. The basidiomycete human pathogen Cryptococcus neoformans changes its morphology; it grows mainly as yeast but during the sexual cycle the fungus forms hyphae and produces sexual spores that are readily disseminated as infectious propagules [45][46][47]. C. neoformans also exhibits pseudohyphal growth, which is advantageous for escape from amoebae, one of its natural predators [48,49]. In the plant pathogenic basidiomycete Ustilago maydis, the yeast form is not virulent; however, the hyphae produced by mating cause disease on plant hosts and transform infected plant tissue into galls [50]. Therefore, morphogenic transitions play major roles during host infection by pathogenic fungi. Dimorphism in zygomycetes, especially in Mucor spp. was studied decades ago; however, our knowledge of its contributions to pathogenesis is limited and the underlying genetics, beyond the involvement of anaerobic/high CO 2 , respiration, and PKA is not well established.
Our study reveals that calcineurin plays a crucial role during the yeast-hyphal growth transition of M. circinelloides. The calcineurin pathway is conserved throughout eukaryotes. Calcineurin is a Ca 2+ /calmodulin-dependent serine/threonine specific protein phosphatase that consists of two subunits: the catalytic A subunit, which has phosphatase activity, and the regulatory B subunit, which binds calcium and the A subunit to activate the enzyme complex. Both subunits are required for enzyme activity. The drugs tacrolimus (FK506) and cyclosporine A (CsA) form complexes with the immunophilins FKBP12 and cyclophilin A, respectively. The protein-drug complexes bind to the hydrophobic interface between the calcineurin A and B subunits to inhibit phosphatase activity [51,52]. We found that FK506 inhibits hyphal growth and instead induces yeast growth in the pathogenic zygomycete Mucor. Gain of function mutations in the calcineurin regulatory B and catalytic A subunit genes were identified that confer resistance to FK506. Furthermore, disruption of the calcineurin regulatory B subunit required for calcineurin activity results in a yeast-locked phenotype, supporting our conclusion that calcineurin regulates the dimorphic transition. Notably, the cnbRD yeast phase locked mutants were less virulent, suggesting that either hyphae are more virulent than yeast or the morphogenic switch is central to pathogenicity of this fungus. In addition, cAMP-dependent protein kinase A activity was found to be elevated during yeast compared to hyphal growth, and also when calcineurin activity was inhibited by FK506 or mutation. We also show that calcineurin is involved in hyphal polarity and spore size dimorphism, which is linked to virulence [53].
Calcineurin is involved in the morphogenesis and virulence of multiple pathogenic fungi (reviewed in [54,55]): in C. neoformans, calcineurin is required for growth at 37uC [56]; in Candida spp., calcineurin functions in antifungal drug resistance/tolerance, survival in serum, and virulence [57][58][59][60]; calcineurin plays a role in morphogenesis in P. brasiliensis [61]; and in Aspergillus fumigatus, calcineurin regulates morphogenesis and thereby pathogenesis [62]. Therefore, calcineurin is a promising antifungal drug target and this study provides a novel foundation to develop approaches to control the emerging fungal infection mucormycosis.

Results
The calcineurin inhibitor FK506 induces M. circinelloides to grow as yeast We found that in Mucor the calcineurin inhibitor FK506 inhibits hyphal growth and drives multi-budded yeast growth ( Figure 1 and Videos S1 and S2). Eight different M. circinelloides isolates (10 3 spores) were each inoculated aerobically on YPD agar media or YPD agar media containing FK506 (1 mg/mL). At 4 days postinoculation, the colonies grown on YPD with FK506 were significantly smaller than those on YPD alone ( Figure 1A). The smaller, compact colonies consisted entirely of yeast form Mucor (data not shown). In liquid culture, with vigorous shaking for aeration, the Mucor isolate CBS277.49 grew exclusively as a mold

Author Summary
Calcineurin is a Ca 2+ /calmodulin-dependent, serine/threonine-specific protein phosphatase. In pathogenic fungi, calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin is an attractive antifungal drug target. The roles of calcineurin in virulence have been established in both major human pathogenic fungi (Candida species, Cryptococcus neoformans/gattii, Aspergillus fumigatus) and in plant pathogenic fungi (Magnaporthe oryzae, Ustilago maydis/hordei). However, the role of calcineurin is currently unknown in pathogenic zygomycetes. We found that the calcineurin inhibitors FK506 and cyclosporine A inhibit the growth of a prevalent zygomycete pathogen, Mucor. This fungus grows as multi-budded yeast under anaerobic conditions, and we have found that even in aerated culture (which without FK506 would result in abundant hyphal growth), Mucor exhibits yeast growth when exposed to FK506. Mucor cnbR mutants that lack the calcineurin regulatory subunit essential for calcineurin activity, are locked in perpetual yeast phase growth, indicating that calcineurin is required for hyphal growth. We further demonstrated that these yeast-locked mutants are attenuated for virulence, illustrating that hyphae or the yeast-hyphal transition are linked to virulence. These findings indicate that: 1) calcineurin governs the yeast/ hyphae morphogenic transition; 2) a link exists between respiration and the calcineurin pathway; and 3) calcineurin inhibitors are attractive anti-mucormycosis drug candidates.
( Figure 1B); however, this isolate exhibited only yeast growth when cultured in the same fashion but in the presence of FK506 (1 mg/ mL). The multi-budded yeast phenotype imposed by FK506 phenocopies Mucor yeast growth under anaerobic/high CO 2 growth conditions ( Figure 1B). This observation indicates that there is a link between respiration/CO 2 sensing and the calcineurin pathway. On the other hand, another calcineurin inhibitor cyclosporine A (CsA) did not induce multi-budded yeast growth, and instead resulted in abnormal, stunted hyphal growth ( Figure S1). Higher concentrations of CsA did not produce yeast growth (data not shown). This result suggests that FK506-specific calcineurin inhibition plays a role in the hyphal-yeast switch. We note that although CsA does stunt hyphal growth, the efficacy may not be sufficient to enforce yeast growth compared to FK506 if levels of the cyclophilin A-CsA complex are insufficient to inhibit all calcineurin activity. This hypothesis is supported by findings that 1) in a sensitized background in which calcineurin activity has been reduced with a sub-inhibitory concentration of FK506, CsA is now fully able to block the dimorphic transition and impose yeast growth ( Figures S2 and 2) in regulatory B subunit mutant CNBR-1 (N125Y, note that the all gain-of-function mutations are designated with capital letters) mutants isolated as being resistant to FK506 (described below), CsA imposes a multi-budded yeast phenotype (See supplemental figure S5).

Identification of calcineurin pathway components in the Mucor genome
We identified calcineurin pathway components in the M. circinelloides genome. Unusually high numbers of calmodulin and calmodulin (CAM) kinase orthologs (nine calmodulins) were identified in the genomes with six CAM kinases Figure S3). Interestingly, the Mucor genome encodes three calcineurin catalytic A subunits along with a single calcineurin regulatory B subunit, FKBP12, and cyclophilin A.
We further examined the three catalytic A subunit (cna) genes and found that the Cna ortholog proteins contain an N-terminal phosphatase domain and a C-terminal regulatory domain containing the calcineurin B subunit binding domain, cyclophilin A-cyclosporine A complex binding domain, FKBP12-FK506 complex binding domain (the domains were determined based on [63]), calmodulin binding domain, and auto-inhibitory domain (Figure 2), further demonstrating that the identified Cna proteins are bona fide calcineurin catalytic A subunits. The sequences of the Cna proteins are distinct, and there is 73% identity shared between CnaA and CnaB; 64% identity between CnaA and CnaC; and 62% identity between CnaB and CnaC. It is interesting to consider how multiple paralogs of the cna genes evolved in the zygomycetes ( Figure S3). In phylogenetic analyses, the three zygomycete species including M. circinelloides, Rhizopus delemar, and Phycomyces blakesleeanus, are conserved on a common branch, including McCnaA, PbCnaA, RdCnaA, and RdCnaB. This observation suggests that the calcineurin A subunit gene in this group may be the more ancestral ( Figure S3).
Gain-of-function mutations in the cnaA and cnbR genes result in hyphal growth in the presence of FK506 FKBP12 is an immunophilin family protein with cis-trans peptide prolyl isomerase activity that serves as the cellular receptor for FK506 and rapamycin [64]. When bound to FK506, FKBP12 binds to the interface between the calcineurin catalytic A and regulatory B subunits, inhibiting phosphatase activity by occluding substrate access to the active site [51]. FKBP12 also binds to rapamycin to inhibit the Tor pathway. Disruption of the gene encoding FKBP12 confers resistance to FK506 and rapamycin (in the absence of FKBP12, FK506 fails to bind calcineurin) [65]. Amino acid substitutions in the calcineurin regulatory B and catalytic A subunit surface that interact with the FKBP12-FK506 complex result in resistance to FK506 [63,66]. Calcineurin FK506 resistant mutants expressing FKBP12 remain rapamycin sensitive. Sensitivity is defined as FK506 enforced yeast growth or rapamycin limiting hyphal growth of the fungus; resistance to FK506 is defined as strains forming hyphae instead of yeast in the presence of FK506, and resistance to rapamycin as strains growing as in non-drug media.
To test whether these molecular principles of FK506 sensitivity and resistance apply in M. circinelloides, we grew wild-type strains (10 3 spores) in solid YPD media containing 1 mg/ml FK506 until resistant mycelial growth emerged out of yeast colonies as In liquid YPD media with vigorous shaking for aeration, wild-type exhibits hyphal growth. When FK506 is present in the media, Mucor grows only as a multi-budded yeast, as is also observed during growth under low oxygen/high CO 2 conditions (isolate grown at the bottom of a flask completely filled with liquid YPD media generating microaerobic/high CO 2 conditions). Representative images with isolate CBS277.49 are shown here. Similar results were observed with other Mucor isolates (data not shown). Scale = 5 mm. doi:10.1371/journal.ppat.1003625.g001 previously described [65]. Through this approach six FK506 resistant spontaneous mutants, MSL11, MSL12, MSL13, MSL14, MSL15, and MSL16 were generated (Table 1 and Figure 3). These FK506 resistant mutants were as rapamycin sensitive as the wild-type parental strain and in contrast to an fkbAD strain lacking FKBP12 that is FK506 and rapamycin resistant. Another mutant strain SM4 has a base substitution in the fkbA gene resulting in a leucine to proline substitution (L91P) in FKBP12 that confers resistance to FK506 but not to rapamycin [65]. Five of six spontaneous mutants remained sensitive to CsA, whereas the MSL16 mutant (CNBR-3) was found to be cross-resistant to CsA (see Figure S6). These observations led us to examine the genes involved in the mode of inhibition by FK506, and we sequenced the cnbR, cnaA, cnaB, and cnaC calcineurin genes as well as the fkbA   Figure S4) and the mutants formed mycelia when the calcineurin inhibitor FK506 (1 mg/mL) is present in the media (The CNBR-3 mutant is shown in Figure S6). The CNBR-1 and CNBR-2, and CNBR-3 mutants are still sensitive to rapamycin. The CNBR-1 and CNBR-2 mutants remain sensitive to CsA, whereas CNBR-3 was found to be CsA cross-resistant ( Figure S5 and S6). The fkbAD mutant and L91P mutants, by contrast, are resistant to FK506 and rapamycin due to the absence or alteration of FKBP12, the target for both drugs, respectively. (C and D) The MSL13 (CNAA-1) and MSL14 (CNAA-2) mutants have mutations in the cnaA gene (see also Figure S7), resulting in amino acid alterations in the binding domains for calcineurin B and for the FKBP12-FK506 complex. Similarly to the CNBR-1, and CNBR-2, the CNAA-1 and CNAA-2 mutants form hyphae in the presence of FK506 but remain sensitive to rapamycin and CsA. doi:10.1371/journal.ppat.1003625.g003 gene to test if any mutations were present in any of these genes in the six FK506 resistant isolates.
Four strains had mutations in only the cnbR gene (Figure 3 and S4). The MSL11 and MSL15 mutants (CNBR-1) contained a point mutation, A496T, which results in an amino acid alteration, N125Y. It is interesting that two independent mutant strains contained an identical mutation in the cnbR gene and these strains displayed yeast growth in the presence of CsA (CsA hypersensitive) ( Figure S5). This mutation may both impair FKBP12-FK506 binding and compromise the stability of the calcineurin A-B complexes to result in CsA hypersensitivity. The MSL12 strain (CNBR-2) contained an insertion of three nucleotides 'CCA' at the 513 th bp of the ORF (513_514insCCA), resulting in an insertion of histidine (H) after 129 th aspragine, Asn129_Gln130insHis. The MSL16 mutant (CNBR-3) contained a point mutation, G487T, which results in the amino acid substitutions, V122F. Interestingly, all of these amino acid alterations are present in the latch region of the calcineurin B regulatory subunit that is known to interact with the FKBP12-FK506 complex and to be involved in phosphatase activity [52,66]. It is likely that the N125Y, N129_Q130insH, and V122F changes alter the interaction between FKBP12-FK506 and the calcineurin B-calcineurin A complex, resulting in resistance to FK506. Interestingly, the V122F substitution may also alter the interaction between cyclophilin-CsA and the calcineurin Bcalcineurin A complex as the MSL16 mutant (CNBR-3, V122F) was found to be cross-resistant to CsA ( Figure S6).
The two other mutant strains had mutations in only the cnaA gene ( Figure 3 and S7). The MSL13 mutant (CNAA-1) has a point mutation, G1514C in the ORF, resulting in an amino acid change S378T, which lies in the amino acid adjacent to a previously known FK506 resistant mutant in calcineurin A in S. serevisiae (W430C) [63]. The MSL14 mutant (CNAA-2) has a point mutation, A1489G in the ORF, resulting in the amino acid substitution, N370D. These mutations occurred in the binding domain for calcineurin B and for the FKBP12-FK506 complex and likely reduce the binding affinity of the FKBP12-FK506 complex to the interface between the calcineurin A and B subunits, resulting in a less efficient inhibition of calcineurin activity by FK506. The fact that two FK506 resistant alleles were isolated in one of the three calcineurin A catalytic subunit (CnaA) suggests that it alone is sufficient to promote hyphal growth when cells are exposed to FK506, as the remaining two calcineurin A catalytic subunits (CnaB and CnaC) in complex with the CnbR regulatory subunit should be inhibited by FKBP12-FK506 under these conditions. The isolation and analysis of these FK506 resistant mutants in calcineurin A and B subunits further support the conclusions that the calcineurin pathway is: 1) conserved, 2) responsible for the dimorphic transition, and 3) the target of FK506 action in M. circinelloides.
Disruption of the regulatory subunit gene cnbR results in a yeast-locked M. circinelloides mutant that is less virulent than wild-type FK506 treatment results in yeast growth in Mucor; therefore, we hypothesized that calcineurin activity is essential to maintain hyphal growth. To test this hypothesis we disrupted the cnbR gene encoding the calcineurin B regulatory subunit as described in the Materials and Methods. The cnbR gene in the wild-type strain MU402 (pyrG 2 , leuA 2 ) was replaced with a cnbRD::pyrG allele, and the gene deletion was confirmed by 59 and 39 junction PCR as well as ORF spanning PCR, and further confirmed by Southern blot analysis to show precise homologous recombination and the absence of any ectopic integration events ( Figures S8 and S9). We generated two independent cnbRD deletion mutants from separate transformations.
The wild-type and cnbRD mutants (10 3 cells) were inoculated in the center of solid YPD medium and the strains were grown for three days at 30uC under normal aerobic conditions. The wildtype formed a large colony consisting of complex mycelia, whereas the cnbRD mutants displayed a compact yeast colony ( Figure 4A). In liquid YPD media, wild-type Mucor grew as a filamentous mold; however, the cnbRD mutants displayed only yeast growth, even in aerobic conditions ( Figure 4B). No impact of FK506 was observed on the cnbRD mutants consistent with a complete absence of the drug target ( Figure 4C). This phenotype of the cnbRD mutants is essentially identical to that of wild-type Mucor grown in the presence of FK506 in aerobic conditions (Videos S2 and S3). The observations that mutation or inhibition of calcineurin enforces yeast growth are the key findings that allow us to conclude that the calcineurin pathway orchestrates the dimorphic switch in Mucor.
To assess the virulence of yeast vs. hyphae and to test the role of the calcineurin pathway in virulence, the cnbRD yeast-locked mutants were compared to both wild-type spores and wild-type yeast cells in animal virulence models ( Figure 5). The R7B (leuA 2 ) strain served as wild-type to exclude the possibility that leucine auxotrophy could generate a biased result, as the cnbRD mutants were generated in the MU402 (pyrG 2 leuA 2 ) strain background (pyrG 2 leuA 2 cnbR::pyrG). Wild-type spores, wild-type yeast, and cnbRD mutant yeast (two independent mutant isolates) were quantified and cohorts of 10 wax moth larvae (a heterologous host system) per strain were infected with an inoculum of 20,000 infectious units. Wild-type spores and wild-type yeast both showed 100% mortality by four days post-infection ( Figure 5A). Wild-type yeast are able to switch into growth as hyphae; thus, it was anticipated that wild-type yeast would be as virulent as wild-type spores. On the other hand, the cnbRD yeast-locked mutants did not kill the larvae after 8 days and were indistinguishable from the PBS control. When 40,000 cells were inoculated, the yeast-locked cnbRD mutants showed a higher level of virulence (60 to 70% mortality by day 4 post-infection), but were still significantly less virulent than wild-type spores (WT vs. cnbRD1, p = 0.00059) ( Figure 5B). These results indicate that: 1) morphogenesis contributes to virulence in this fungus; 2) the calcineurin pathway is involved in virulence, and 3) calcineurin inhibitors have promise as antifungal drugs for zygomycete infections.
That hyphae are more virulent than yeast, or alternatively that ability to transition between yeast to hyphae is a key virulence factor as observed in Candida albicans [40], is further supported by observations of tissues from infected animal host models ( Figure  S10). In a wax moth mucormycosis system, wild-type mainly presented as the hyphal form, whereas the yeast-locked mutants were exclusively as yeast. In a murine mucormycosis system, we found that the majority of an M. circinelloides fungal burden was present in the brain as hyphae.
cAMP-dependent protein kinase A activity is elevated during yeast growth in anaerobic/high CO 2 conditions, in the presence of FK506, and in the cnbRD mutants Yeast growth can be induced by elevated levels of CO 2 [15,16]. Carbon dioxide is converted into bicarbonate ions (HCO 3 2 ) by carbonic anhydrase (reviewed in [67] and references therein). Subsequent activation of adenylyl cyclase by CO 2 , HCO 3 2 , or both occurs, resulting in the production of cAMP. cAMP then binds to the regulatory subunit of protein kinase A (PKAR) and the protein kinase A (PKA) catalytic subunits are released to result in activation. Previous studies suggested that PKA may play a major role during yeast growth, given that the PKA genes are over-expressed during yeast growth, and the regulatory subunit gene (pkaR) that inhibits PKA activity is expressed at a higher level during the yeast to hyphal transition [25,27,28]. We further confirmed that Mucor grows as yeast in the presence of high concentrations of bicarbonate in the media. At 50 mM bicarbonate, Mucor exhibited both hyphal and yeast growth; at 75 mM, Mucor grew only as a yeast ( Figure 6A).
We adapted a PKA activity assay used in other studies of Mucor morphogenesis to test how PKA activity is differentially regulated during yeast and hyphal growth [27,28,68]. Due to the nature of this assay performed in the presence of cAMP, the maximum possible PKA activity was measured. Crude cell extracts (0.5 mg total protein) were prepared and PKA activity was measured by using the PKA specific substrate kemptide as previously described [27,68]. Compared to hyphal growth, PKA activity was elevated approximately five-fold during yeast growth in high CO 2 growth conditions ( Figure 6B). During yeast growth enforced by FK506, PKA activity was higher. In the two independent cnbRD yeastlocked mutants, PKA activity was also higher than in wild-type ( Figure 6B). These results demonstrate that 1) higher activity of PKA is associated with, and may be necessary for, yeast growth; and 2) calcineurin and PKA may play antagonistic roles during the yeast-hyphal dimorphic transition. It is noteworthy that the M. circinelloides genome encodes four PKA regulatory subunits and their subcellular localization may generate compartmentalized PKA activity [26][27][28]. In the non-dimorphic zygomycete R. delemar, PKA activity was also elevated by FK506 treatment, and the same result was observed in the basidiomycete pathogen C. neoformans ( Figure S11).

Calcineurin catalytic A subunits are expressed differentially during hyphal and yeast growth
We tested whether the three catalytic A subunit genes (cnaA, cnaB, and cnaC) are differentially regulated during the dimorphic transition. Initial northern blots showed that the probes for each cna gene were cross-reactive due to sequence similarity (data not shown); therefore, we performed quantitative RT-PCR with gene specific primers to specifically examine expression of each individual gene. In RT-PCR analyses, we found that all three cna genes were expressed, indicating that none were pseudogenes (data not shown). The PCR products from cDNAs obtained for each gene were sequenced to re-annotate the genes. Based on the ORF sequences, we designed a pair of specific RT-PCR primers across two exons of each cna gene. The actin gene was used for normalization.
The expression levels of the cna genes were evaluated using ct values from RT-PCR with hyphal growth conditions as a control. We found that cnaC is expressed at higher levels during On YPD solid media, the cnbRD mutants lacking all calcineurin activity (CnbR is the common subunit partner for the three catalytic subunits CnaA, CnaB, and CnaC) produce a small compact colony; however, wild-type forms a complex mycelial mat. (B) In liquid media with vigorous shaking for aeration, wild-type grows as a filamentous/hyphal fungus, whereas the cnbRD mutants only exhibit multi-budded yeast growth as observed in wild-type grown in the presence of FK506 or anaerobic/high CO 2 conditions. (C) The two cnbRD mutants were serially diluted and spot-inoculated on YPD or YPD with FK506 (1 mg/L). yeast growth under microaerobic conditions but at lower levels during hyphal growth. Interestingly, during yeast growth driven by FK506, cnaC is also overexpressed (Figure 7). The expression levels of cnaA and cnaB were moderately reduced during the dimorphic switch. These results suggest that CnaC plays a specific role during the yeast growth phase and could therefore be less sensitive to inhibition by FK506, possibly as the result of overexpression in molar excess above FKBP12 levels. In parallel studies, Drs. Praveen Juvvadi and William Steinbach at Duke University found that M. circinelloides cnaC partially complements the radial growth defect of the A. fumigatus calcineurin catalytic A subunit (cnaA) mutant and also that MuCnaC-GFP localizes to septa, as does AfCnaA (Juvvadi P. R. et al., manuscript in preparation). M. circinelloides hyphae are coenocytic (aseptate), but during yeast growth or pseudohyphal growth, septa are formed; thus, CnaC function may be associated with septum formation.
Disruption of the cnaA gene results in a hypersensitivity to calcineurin inhibitors and SDS, the production of larger spores, and a loss of hyphal polarity Of the three cna genes found in Mucor, homologs for the cnaA gene were found in all three of the zygomycete genomes analyzed ( Figure S3), indicating that the cnaA gene may be the more ancestral calcineurin catalytic A subunit gene. Therefore, we chose to disrupt the cnaA gene to further test the roles of calcineurin in Mucor. A disruption allele consisting of the pyrG gene flanked by ,1 kb of the 59 upstream and 39 downstream untranslated regions of the cnaA gene were introduced as described in the Materials and Methods. Gene replacement by recombination was confirmed by 59 and 39 junction PCR, ORF spanning PCR, and Southern blot (Figures S12 and S13). Two independent cnaAD::pyrG mutants were obtained from separate transformations.
Interestingly, the cnaAD mutants exhibited much higher sensitivity to FK506 and CsA compared to wild-type ( Figure 8A). Sensitivity was assessed based on the ability of the drugs to inhibit growth of the strains tested; for example, on YPD medium supplemented with FK506 (0.025 to 1 mg/ml), the two cnaAD mutants displayed significantly reduced growth compared to wildtype. Both cnaAD mutants also exhibited hypersensitivity to CsA. In liquid YPD with vigorous aeration, more abundant yeast growth was observed in the cnaA mutants compared to wild-type. Notably, the cnaAD mutants exhibited only yeast growth in the  20,000 wild-type spores, wild-type yeast, or the cnbRD yeast-locked mutants were inoculated into wax moth larvae. Infection with wild-type spores or yeast resulted in 100% mortality by day 4 post-infection; however, the cnbRD yeast-locked mutants were significantly less virulent compared to wild-type spores or yeast cells. (B) At an inoculum of 40,000 cells, the cnbRD mutants exhibited 60 to 70% mortality by day 2 post-infection, whereas wildtype spores showed 100% mortality. doi:10.1371/journal.ppat.1003625.g005 presence of 0.1 mg/ml FK506 in the media, which is a concentration insufficient to inhibit hyphal growth and induce yeast growth in wild-type ( Figure 8B). We explain these observations as resulting from the absence of one catalytic A subunit out of the three expressed (the mutants still have intact cnaB and cnaC genes), leading to a relative decrease in calcineurin, similar to haplo-insufficiency in S. cerevisiae [69]. The cnaAD mutants were also more sensitive to SDS, indicating that calcineurin may be involved in cell wall integrity ( Figure 8C) as observed in Candida species [57,70]. Mucor was largely resistant to Congo red and calcofluor white and there was no significant difference between wild-type and cnaA mutants with either (data not shown).
We found that the cnaAD mutants produce larger spores than the wild-type (WT: 12.0062.94 mm; cnaAD1: 16.0864.04 mm; cnaAD2: 16.0263.70 mm) ( Figure 9A and B). The differences in spore size between the wild-type and the two cnaAD mutants are significant based on a two sample independent t-test (p,0.0001 in both WT vs. cnaAD1 and WT vs. cnaAD2, N = 80). The cnaA mutant spores were multinucleate as are wild-type spores. This larger spore size is an intriguing phenotype because spore size is a virulence factor as shown in our previous study, wherein larger spores were more virulent than smaller spores [53].
To test if the enlarged spore phenotype conferred by mutations in the cnaA gene might contribute to virulence, we infected wax moth larvae with 5,000 spores of wild-type or the cnaAD mutants. Ten larvae were used for each strain. At the given inoculum, wildtype showed a moderate level of virulence; however, the two cnaA mutants were significantly more virulent compared to the wildtype ( Figure 9C). The cnaAD mutants also formed hyphae in hosts similar to wild-type. (Figure S14). The experiments were performed independently three times with similar results. As observed in our previous study [53], in the interaction with macrophages, the larger cnaAD mutant spores produced a germ tube inside of macrophages, whereas wild-type remained as spores without germ tube emergence at 3.5 hours of co-culture ( Figure  S15), indicating that the mutants possibly avoided the innate host immune system by sending out germ tubes earlier and more efficiently than the wild-type. This suggests that calcineurin is involved in spore size control as a negative regulator and may therefore also contribute to virulence, although other calcineurin-CnaA-dependent functions may also contribute.
The cnaAD mutations also confer abnormal hyphal polarity. To carefully examine hyphal growth of the cnaAD mutants, we applied a time course imaging technique with a microscope equipped with an automatic shutter system. Wild-type and cnaAD mutant spores were placed on YPD agar media, and images were captured every 30 seconds. When the cnaAD mutants germinated, the germ tubes exhibited a tip-splitting phenotype and abnormal branching, whereas wild-type germ tubes elongated in a single direction with no tip-splitting ( Figure 10 and Videos S4 and S5). This phenotype of the cnaAD mutants indicates that CnaA is necessary to maintain a single hyphal polarity in Mucor. A role for calcineurin in hyphal polarity is conserved in other filamentous fungi, including Neurospora crassa and A. fumigatus [62,71], indicating that this is a general role of calcineurin.

Discussion
Morphogenic transitions (for example, yeast to hyphae and vice versa) are common in fungi and evolved in multiple fungal lineages. Many well known fungal pathogens change their morphology in response to their environments and morphogenesis is linked to their virulence. Human fungal pathogens encompass ascomycetes, basidiomycetes, and zygomycetes [72]. In ascomycetes and basidiomycetes, the relationships between dimorphism and pathogenicity are relatively well established. However, whether dimorphism is associated with virulence in zygomycetes was not known. Our study has revealed how the dimorphic transition is regulated and linked to virulence and how the calcineurin pathway directs this process in the human pathogenic zygomycete M. circinelloides.
Several zygomycete species, especially those belonging to the order Mucorales, are known to cause the lethal fungal infection mucormycosis. These species include Mucor spp., Rhizopus spp., Rhizomucor spp., Cunninghamella spp., and A. trapeziformis [1][2][3]. Unlike ascomycetous and basidiomycetous fungal pathogens, the current status of research into zygomycete pathogens is in its infancy. A limited ability to conduct genetics studies is one of the barriers to advancement in our understanding of zygomycete pathogens. M. circinelloides is one of the best developed systems for genetics and molecular biology among zygomycete species. Therefore, Mucor provides a foundation from which to understand mucormycosis.
Mucor is a dimorphic fungus and the morphogenic transition is known to occur in response to environmental conditions, especially low levels of oxygen and high levels of carbon dioxide. Several other studies have implicated protein kinase A in this response [25][26][27][28][29][30][31]. Other factors known to be involved predominantly alter mitochondrial functions. Our study revealed that the calcineurin pathway is a key regulator of this developmental process. (Figure 1). Previously, we found that FKBP12 mutants are resistant to FK506 (resistance is defined as strains forming hyphae instead of yeast in the presence of FK506) [65]. FKBP12 is a member of the immunophilin family of proteins, which have cis-trans peptidylprolyl isomerase activity [64]. When bound to FK506, FKBP12 binds to the interface between the calcineurin catalytic A and regulatory B subunits, inhibiting phosphatase activity by occluding substrate access to the active site [51]. FKBP12 also binds to rapamycin to inhibit the Tor pathway. In other fungi, disruption of the gene encoding FKBP12 confers resistance to FK506 and rapamycin (in the absence of FKBP12, FK506 fails to bind calcineurin). Similar results are observed in Mucor; a splice site mutant (adenine into guanine at the 316 th nucleotide), Leu91Pro In the presence of low concentrations of FK506 (0.1 mg/L), growth of the cnaAD mutants was significantly impaired compared to the wild-type, which formed a readily visible colony after only 1 day. (B) In liquid culture with a low concentration of FK506 (0.1 mg/mL), at which wild-type grows as hyphae, the cnaAD mutants grew as multi-budded yeast cells. Even in culture without FK506, the cnaAD mutants exhibited a mixture of yeast and hyphae compared to wild-type, which was exclusively hyphal. Scale = 20 mm. (C) The cnaAD mutants were more sensitive to SDS in YPD media than wild-type, suggesting that the cnaAD mutants fail to have compromised cell wall integrity. The percentage concentration (v/v) of SDS is displayed in the panel. doi:10.1371/journal.ppat.1003625.g008 substitution mutant, and FKBP12D null mutants are all resistant to FK506 [65].

FK506 treatment imposes yeast growth in Mucor
In this study, we found that the N125Y, N129_Q130insH, and V122F alterations in the latch region of the calcineurin B regulatory protein confer resistance to FK506 (Figure 3 and S4). A previous site-directed mutagenesis study documented that the latch region is essential for immunophilin-immunosuppressant complex docking [52]. These mutations in the CnbR subunit of Mucor may prevent or limit docking of FKBP12-FK506 onto the calcineurin complex to result in less inhibition of calcineurin. Similar results has been observed in C. neoformans where a two amino acid insertion in the latch area of calcineurin B similarly confers resistance to FK506 [66]. In addition, the N370D and S378T mutations in the calcineurin B and FKBP12-FK506 binding domain of the CnaA catalytic subunit (Figure 3 and S7) confer resistance to FK506 and lead to the conclusion that Mucor has a conserved calcineurin pathway. Furthermore, the calcineurin B regulatory subunit gene disruption mutants only exhibited yeast growth, even during vigorous aeration conditions and there was no impact of FK506 on these strains that lack calcineurin ( Figure 4). These observations all support the conclusion that calcineurin is a key factor in the dimorphic yeast to hyphae transition in Mucor.
Calcineurin has been suggested as a candidate antifungal drug target in many other pathogenic fungi, including Candida spp., C. neoformans, A. fumigatus, Magnaporthe oryzae, and U. maydis (reviewed in [55]). The observation that calcineurin inhibitors block hyphal growth in zygomycete pathogens parallels findings in other fungal pathogens. That transplant patients receiving FK506 treatment have a lower incidence of mucormycosis contributes to validate calcineurin as a potential antifungal drug target [73]. FK506 displays synergistic effects when combined with other antifungal agents, including azoles and echinocandins in C. albicans (reviewed in [74]). In addition, calcineurin inhibitors exhibit an in vitro synergistic inhibition on zygomycete growth [75][76][77]. A recent study suggests that combination therapy with posaconazole and FK506 is efficacious in both a Drosophila heterologous host model and a cutaneous mucormycosis murine model system [78]. We found that yeast-locked cnbRD mutants are not as virulent as wildtype in a heterologous wax moth model system ( Figure 5). One possibility is that hyphae are a more virulent form of Mucor compared to yeast. However, although the wild-type existed as hyphae and the yeast-locked mutants existed as yeast inside hosts ( Figure S10), we cannot rule out the possibility that the ability to transition between yeast and hyphae is a major factor in virulence during host infections as observed in C. albicans [40]. Currently there are no known hyphal-locked mutants in Mucor that could be used to test these hypotheses further. This observation overall supports two important conclusions: first, dimorphism is linked to virulence in this fungal species and second, calcineurin inhibitors may be an effective way to control mucormycosis.
However, because the calcineurin pathway is conserved throughout eukaryotes, including humans, calcineurin inhibitors have their own inherent risks as antifungal drugs, including for example host immune suppression. In humans, calcineurin dephosphorylates the cytoplasmic subunit of the transcription factor nuclear factor of activated T-cells (NFATc) and promotes its nuclear localization to induce interleukin 2 (IL-2) and stimulate T cell proliferation [79]. Thus, calcineurin inhibitors are immunosuppressive drugs. It is possible that the effects of calcineurin inhibitors as immunosuppressants will overpower their antifungal activities. This situation was also observed in our murine host experiments, in which FK506 treatment detrimentally affected mice infected with Mucor ( Figure S16). Therefore ideal calcineurin Figure 9. cnaAD calcineurin A subunit mutants produce more virulent and larger spores than wild-type. (A) The cnaAD mutants and wild-type produced multinucleate spores (stained with DAPI), and the mutant spores were larger than those of wild-type. DIC: differential interference contrast, DAPI: nuclei. (B) Wild-type produced spores with a size of 12.062.95 mm, whereas the cnaAD1 and cnaAD2 strains produced significantly larger spores with a size of 16.164.04 or 16.063.70 mm, respectively. The difference in spore size between wildtype and the cnaAD mutants was statistically significant (p,0.0001, N = 80). (C) The larger spores of the cnaAD mutants were more virulent than wild-type at the inocula tested (N = 5,000), suggesting that calcineurin is involved in spore size determination and providing further evidence that spore size is linked to the virulence of this fungus. doi:10.1371/journal.ppat.1003625.g009 inhibitors would be FK506 analogs that are less immunosuppressive to the host but which remain active against fungal calcineurins.
Mucor encodes three calcineurin catalytic A subunits (Figures 2  and S3). The presence of the triplicated cna genes might have been derived from individual gene duplication events, which is supported by phylogenetic analyses and the presence of repetitive sequences linked to the cna genes ( Figures S2 and S17). However, in Mucor, synteny around the three cna genes was not conserved ( Figure S17), indicating that the duplication of the cna genes may have resulted from individual gene duplication events or segmental gene duplications that occurred earlier and that flanking synteny has decayed with evolutionary time. The presence of the same repetitive elements up-and downstream of the cna genes supports the hypothesis that individual gene duplication events may have produced the three versions of the cna genes in Mucor. However, in R. delemar, the triplicated copies of the cna genes arose via a different trajectory, involving a recent whole genome duplication and then a segmental gene duplication. Interestingly, the non-pathogenic Mucorales species Phycomyces has only a single cna gene ( Figure S3). These observations indicate that different evolutionary trajectories gave rise to genes linked to virulence during speciation in the Mucorales order.
The cnaA gene is conserved in the three Mucorales species, and this gene might therefore have a conserved ancestral role in this fungal lineage. The Mucor cnaAD mutants display several interesting phenotypes: an abnormal polarity during hyphal growth, larger spore production, hypersensitivity to calcineurin inhibitors, and cell wall defects (Figures 8, 9, and 10, and Videos S3 and S4). The abnormal polarity phenotypes include tip-splitting and atypical branching patterns. In several other fungal species, calcineurin is involved in hyphal polarity in accord with our observations in Mucor [62,71,80]. A remarkable phenotype of the cnaAD mutants is the production of larger spores (Figure 9). In a Figure 10. Hyphal growth is altered in cnaAD mutants. (A) Wild-type spores first grow isotropically and then polarity is established to initiate germ tube emergence. During hyphal growth, polarity is maintained properly in order to grow directionally. (B) Compared to wild-type, the cnaAD mutants exhibited a shorter isotropic growth phase due to their larger initial spore size (the cnaAD mutants germinated at 50 minutes after inoculation, whereas wild-type started germination at 75 minutes after inoculation, as shown in the figure) [53]. cnaAD mutant hyphae undergo tipsplitting (solid red arrows), indicating that hyphal polarity is not properly maintained during mycelial growth. The cnaAD mutants also show an abnormal hyphal branching pattern (open red arrows). The numbers on the figure indicate the time (in minutes) after inoculation. Scale = 20 mm. doi:10.1371/journal.ppat.1003625.g010 previous study, we showed that larger spore size is linked to higher virulence in Mucor. Accordingly, the larger spores of the cnaAD mutants were also found to be more virulent in the heterologous host model and macrophage model (Figures 9 and S15), further supporting the hypothesis that fungal cell gigantism is involved in virulence as observed in titan/giant cells of C. neoformans and multinucleate spherules of Coccidioides immitis/posadasii [53,[81][82][83][84]. This observation is at one level paradoxical because we suggest calcineurin as a target of antifungal drugs. However, we found that the growth of the cnaAD mutants was more sensitive to FK506 and CsA, and that the cnaAD mutants also exhibit cell wall defects (Figure 8). Taken together with the finding that cnbRD mutants that should lack all calcineurin activity are attenuated in virulence, these results indicate that calcineurin inhibitors remain attractive antifungal drug candidates.
Calcineurin regulates many cellular processes in eukaryotes, including T-cell activation, polarized growth of neurites, long-term memory transitions, signaling in cardiac hypertrophy in humans as well as morphogenesis and virulence in fungi [55,79,[85][86][87]; however, to our knowledge, a connection between respiration and calcineurin has not been previously described. Our finding that calcineurin negatively regulates protein kinase A (PKA) activity is also novel (Figures 6 and S11). An antagonistic relationship between calcineurin and PKA has been observed in U. maydis and S. cerevisiae [88,89]. Our study verifies that calcineurin activity is required for hyphal growth and PKA activity is correlated with yeast growth. Further, calcineurin activity might attenuate PKA activity in Mucor. Similar results were observed in two other pathogenic fungi, C. neoformans and R. delemar; thus, this antagonistic interconnection between calcineurin and PKA may be a conserved functional circuit in several fungal species. It is not clear if calcineurin directly acts to dephosphorylate PKA to control its activity or indirectly regulates PKA activity, for example, by regulation at the transcriptional level or via other proteins. Alternatively, calcineurin and PKA may share common targets (e.g. Crz1) for dephosphorylation and phosphorylation, respectively ( Figure 11). Future investigation of this signaling network will be a promising venture.
In conclusion, our finding that calcineurin orchestrates the dimorphic transition in Mucor is a significant advance since the first finding that CO 2 /low oxygen was the key factor [15][16][17]. Another key major advance of this study is that dimorphism is linked to virulence in the zygomycete pathogen Mucor. This study on morphogenesis and pathogenicity in the emerging zygomycete fungal pathogens will foster development of anti-mucormycosis targets for novel treatment options.

Strains and growth conditions
Strains and plasmids used in this study are listed in Table 1. M. circinelloides strains were grown on yeast and dextrose agar (YPD, 10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, 2% agar) or yeast peptone glucose (YPG, 3 g/L yeast extract, 10 g/L peptone, 20 g/L glucose, 2% agar, pH = 4.5) media for spore production at room temperature (24uC) or at 30uC in the light. YPD was used for liquid culture with vigorous shaking for aeration at 30uC. For anaerobic/high CO 2 conditions, 500 mL flasks were completely filled with liquid YPD media that had been sonicated to eliminate residual O 2 gas. The spores of experimental strains (10 7 spores) were then inoculated on the bottom of the flasks. The flasks were sealed with parafilm and placed in an incubator at 30uC without shaking. Plasmids used in this study were maintained in the Escherichia coli DH5a-T1R strain (Invitrogen, Carlsbad, CA) and manipulated as previously described [90]. All microbial strains were handled under appropriate Biosafety Level 2 conditions (BSL2). All chemicals for media, buffer, and supplements were purchased from Difco Laboratories (Sparks, MD) or Sigma-Aldrich (Saint Louis, MO) unless otherwise stated.
Liquid YPD or YPD agar plates were supplemented with FK506 (1 mg/mL) or cyclosporine A (100 mg/mL). Various concentrations of FK506 were also used (0.025 to 1 mg/mL). Identification of calcineurin pathway components, annotation of the calcineurin catalytic A subunit genes in M. circinelloides, and phylogenetic analysis Three available zygomycete genomes were examined, including M. circinelloides (http://genome.jgi-psf.org/Mucci1/Mucci1.home. html), R. delemar (http://www.broadinstitute.org/annotation/ genome/rhizopus_oryzae/MultiHome.html), and P. blakesleeanus (http://genome.jgi.doe.gov/Phybl2/Phybl2.home.html). To assess the calcineurin components, including the calcineurin catalytic A and regulatory B subunits, FK506 binding protein 12 (FKBP12), cyclophilin A, calmodulin, and calmodulin-dependent protein kinase (CAM kinase), we applied a BLAST search by using S. cerevisiae and C. neoformans calcineurin pathway proteins. Proteins in the zygomycete genomes identified via a BLAST with S. cerevisiae and C. neoformans proteins were re-BLASTED against the S. cerevisiae and C. neoformans genomes. When the proteins in the zygomycete genomes returned the S. cerevisiae and C. neoformans counterpart proteins as the best hits, we defined them as the calcineurin components in the zygomycete genomes.
Fungal mass was lyophilized, and total RNA was extracted from the lyophilized fungal mass using the Ambion RiboPure-Yeast Kit (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from the extracted total RNA using the AffinityScript qPCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA). The ORFs of the three cna genes were each amplified with a pair of primers: JOHE23748 and JOHE23750 for cnaA; JOHE23751 and JOHE23753 for cnaB; JOHE23754 and JOHE23756 for cnaC. Primers used in this study are listed in Table S1. PCR products obtained were directly sequenced with the primers above and additionally with JOHE20751 for cnaA, JOHE23765 and JOHE23766 for cnaB, and JOHE23767 and JOHE23768 for cnaC.
Deduced amino acid sequences of the cna genes were aligned with CLUSTALW, along with the calcineurin catalytic A subunit protein sequences from R. delemar, P. blakesleeanus, S. cerevisiae, Aspergillus nidulans, C. neoformans, and Homo sapiens. Phylogenies were constructed using a PhyML 3.0 software [91], which allowed phylogenies to be inferred and levels of support ascertained. Phylogenetic trees were drawn with the Dendroscope program [92] with aligned sequences.
Generation of spontaneous mutants that are resistant to FK506 and sensitive to rapamycin  (Table 1) in the center of YPD solid medium containing FK506 (1 mg/mL), as described before [65]. Between 5 days and 2 or 3 weeks of growth, some of the yeast colonies showed resistant mycelial outgrowths, from which spores were collected and passaged on FK506-containing solid medium until homokaryotic mycelial growth was observed. Spore Figure 11. Illustration of roles of calcineurin in dimorphism and virulence of Mucor. Calcineurin is necessary for hyphal growth and negatively regulates spore size. Calcineurin is also involved in cell wall integrity. Calcineurin activity is inhibited by two immunophilin-drug complexes, FKBP12-FK506 and CypA-CsA, resulting in yeast growth or abnormal hyphal growth, respectively. On the other hand, during yeast growth, cAMP-dependent protein kinase A (PKA) activity is increased. I) Inhibition of calcineurin activity by FK506 or mutation of the regulatory B subunit results in elevated PKA activity, indicating that calcineurin negatively regulates PKA activity, either via direct inhibition of PKA or indirectly through other factor(s). II) Alternatively, PKA and calcineurin may share common target proteins and function. FKBP12: FK506 binding protein 12, CypA: cyclophilin A. doi:10.1371/journal.ppat.1003625.g011 suspensions were collected and tested for rapamycin resistance on YPD solid medium containing rapamycin (100 mg/mL). Total genomic DNA was isolated from the mutants and the genes encoding fkbA, cnbR, cnaA, cnaB, and cnaC were sequenced.
Disruption of cnbR, the calcineurin regulatory B subunit gene, and cnaA, a calcineurin catalytic A subunit gene To disrupt the cnbR gene, we constructed a disruption allele containing the pyrG gene flanked by ,1 kb of the 59 and 39 untranslated regions of the cnbR gene via overlap PCR. The 59 region was amplified with primers JOHE22226 and JOHE22227, and the 39 region was amplified with primers JOHE22230 and JOHE22231 from the genomic DNA of wild-type M. circinelloides strain CBS277.49 (the genome sequence reference strain). The pyrG gene was also amplified with primers JOHE22228 and JOHE22229 from CBS277.49 genomic DNA. The three fragments were then subjected to an overlap PCR with nested primers JOHE22236 and JOHE22237 to isolate a disruption allele as described [93]. The cnbRD::pyrG cassette was purified and cloned into plasmid pCR2.1-TOPO following the manufacturer's instructions (Invitrogen, Carlsbad, CA). The resulting plasmid, pCnbR-KO, was used to generate a greater amount of disruption construct DNA via PCR.
Strain MU402 (leuA 2 , pyrG 2 ) was transformed with the obtained disruption cassette to disrupt the target cnbR gene, and transformation was carried out as described previously [94]. In brief, protoplasts (confirmed by lysis in water) were obtained from 2.5610 8 germinated spores of strain MU402 (pyrG 2 , leuA 2 ) [95] by incubation with 0.03 unit/mL chitosanase RD (US Biological, Marblehead, MA) and 1 mg/mL lysing enzymes (L-1412; Sigma-Aldrich, Saint Louis, MO) at 30uC for 90 min. Protoplasts were incubated with 5 mg of DNA and electroporation was performed in 0.2 cm cuvettes (pulse at 0.8 kV, 25 mF, and 400 Ohms). pyrG + transformants were selected on MMC medium (1% casamino acids, 0.05% yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose) pH 3.2, supplemented with 0.5 M sorbitol [95]. Mucor is coenocytic (aseptate), and therefore, an intensive selection procedure is required to obtain progeny with a homozygous karyotype. Thirty-nine transformants were obtained from eight independent transformations. The transformants were then grown in MMC selective medium for several vegetative cycles to increase the proportion of transformed nuclei. Spores of the transformants were spread onto MMC medium with uridine (200 mg/L) and MMC medium without uridine to determine the ratio between pyrG + and pyrG 2 progeny. Once the ratio reached 1:1, genomic DNA from the transformants was extracted and used as a template for 59 and 39 junction PCRs to identify transformants in which homologous replacement of the cnbR locus with the pyrG allele had occurred. We found two independent transformants that contained the cnbRD::pyrG allele. Then, the two transformants underwent intensive vegetative selection cycles until all progeny were pyrG + , and 59 and 39 junction PCRs then allowed us to confirm the disruption of the cnbR gene ( Figure S8). JOHE22226 and JOHE37644 were used to amplify the 59 junction and JOHE22231 and JOHE37645 to amplify the 39 junction. Primers JOHE22226 and JOHE22231 are specific to the 59-and 39 regions outside of and upstream and downstream of the disruption cassette, respectively and were used for ORF spanning PCR. Primers JOHE37644 and JOHE37645 are specific to the marker gene pyrG. Primers For Southern blot analysis, primers JOHE22237 and JOHE39531 were used to amplify the probe ( Figure S9).
Disruption of the cnaA gene was performed with the primers listed in Table S1. Thirty-seven transformants were obtained from eight independent transformations, and then screened for homologous recombination as described above. The cnaA gene disruption was confirmed by the same procedure described above. Primers JOHE26840 and JOHE37644 were used to amplify the 59 junction and primers JOHE26845 and JOHE37645 to amplify the 39 junction ( Figure S11). Primers JOHE26840 and JOHE26845 were also used for ORF spanning PCR. The plasmid containing the cnaA disruption cassette in pCR2.1-TOPO was designated pAL1-1 (Table 1). Primers JOHE26847 and JOHE39530 were used to amplify the probe for Southern blot analysis ( Figure S12).

Virulence test
Wild-type and the cnaAD mutant spores were obtained from cultures on solid YPG media that were incubated for 4 days in the light. Sterile deionized water was then added to the media and spore suspensions were collected by scraping with a spreader. For the wild-type yeast, spores were inoculated at the bottom of a 500 mL flask completely filled with liquid YPD media and the flask was incubated without shaking to render the growth conditions anaerobic. The cnbRD mutant yeast were grown on YPD agar (pH = 6.5) for one day at 30uC and yeast colonies were scraped and resuspended in sterile deionized water. All inocula were washed twice with sterile PBS and quantified with a hemocytometer. Inocula in PBS were injected into wax moth larvae, and the survival rate of the host was monitored. The significance of the mortality rate data was evaluated by using Kaplan-Meier survival curves with the PRISM statistics software (GraphRad Software, Inc.).

cAMP-dependent protein kinase A activity assay
Each strain of Mucor, R. delemar RA99-880, and C. neoformans H99 was grown in appropriate conditions. Crude protein extracts were obtained with radioimmunoprecipitation (RIPA) lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) containing PMSF, sodium orthovanadate (a phosphatase inhibitor), and protease inhibitor cocktail solutions as recommended by the manufacturer. In brief, total fungal cells were briefly washed with PBS and soaked in the RIPA buffer with microbeads (426 to 600 mm, Sigma-Aldrich, Saint Louis, MO). Each sample was vigorously shaken in a bead beater 5 times for 1 minute each, rested for 5 minutes at 4uC, and centrifuged for 15 minutes at 12,000 rpm. Supernatants containing crude protein extracts were transferred into a new tube. Total protein was quantified with Bradford solution (BioRad, Hercules, CA) using a standard curve with bovine serum albumin. Protein kinase A activity of the crude protein extracts was measured with the SignaTECT cAMP-Dependent Protein Kinase (PKA) Assay System (Promega, Madison, WI) following the manufacturer's instructions. Three independent experiments were performed with consistent results.

Real-time PCR
To determine the level of expression of the cnaA, cnaB, and cnaC genes during hyphal and yeast growth, quantitative RT-PCR assays were performed. Based on the ORF sequences, we designed pairs of specific RT-PCR primers across two exons of each cna gene: JOHE24019 and JOHE24020 for cnaA; JOHE24021 and JOHE24022 for cnaB; JOHE24023 and JOHE24024 for cnaC and JOHE24075 and JOHE24076 for actin. qRT-PCR was performed using the Brilliant SYBR Green QPCR Master Mix (Agilent Technologies), and the expression of the actin gene was used for normalization. The expression levels of the cna genes were evaluated by ct values from RT-PCR. Multiple independent experiments were performed with concordant results.

Microscopy
Spores and hyphae were observed with a Zeiss Axioskop 2 Plus equipped with an AxioCam MRm camera (Carl Zeiss Inc., Thornwood, NY). To analyze nuclei, spores were fixed with 3.7% formaldehyde in 50 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100 (v/v). The specimens were then mounted on a glass slide with ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA).
Time lapse analyses for the germination of wild-type and cnaA mutants were performed by using a Zeiss Axio Observer Z1 microscope system (Carl Zeiss Inc., Thornwood, NY) with an Opto-electronically motorized XY stage, Pecon XL S1 incubator, and Coolsnap ES2 high-resolution CCD camera, which are housed in the Duke University Light Microscopy Core Facility (LMCF). The images were sequentially obtained every 30 seconds and combined as a movie (30 frames per second) by using MetaMorph 7.6.5 (Molecular devices Inc., Sunnyvale, CA). Figure S1 Phenotypes of M. circinelloides grown in the presence of calcineurin inhibitors FK506 or cyclosporine A (CsA). FK506 induces yeast growth; however, exposure to the other calcineurin inhibitor CsA resulted in abnormal hyphal growth instead of inducing yeast growth. FK506-specific inhibition of calcineurin may drive Mucor to grow as yeast. Alternatively, CsA may not be fully functional in this organism, for example, CsA less efficiently inhibits calcineurin (See text for detailed discussion). Scale = 10 mm.  Figure S5 An N125Y substitution in the calcineurin regulatory B subunit results in resistance to FK506 but hypersensitivity to CsA. (A) The CNBR-1 (N125Y) mutant is resistant to FK506, forming hyphal growth in the presence of FK506 (1 mg/L), and under this condition the wild-type only grows as yeast. However, the CNBR-1 mutant displaysed higher sensitivity to CsA (100 mg/ L) compared to the other CNBR mutants: the CNBR-1 mutant grew much slower than the CNBR-2 and fkbAD mutants. The fkbAD mutant was also resistant to rapamycin, whereas CNBR-1 and CNBR-2 mutants were sensitive. (B) When observed under the microscope, the two CNBR-1 mutants exhibited yeast growth in the presence of CsA (100 mg/L), whereas the wild-type displayed abnormal hyphal growth. Scale = 20 mm. (TIF) Figure S6 A V122F substitution in the calcineurin regulatory B subunit confers cross-resistance to FK506 and CsA. The CNBR-3 (V122F) mutant is resistant to both of FK506 and CsA, whereas the other calcineurin mutants, CNBR-1, fkbAD, and SM4 (L91P) mutants are resistant to FK506 but sensitive to CsA. The fkbAD mutant is resistant to rapamycin, whereas the SM4, CNBR-1, and CNBR-3 mutants are sensitive. (TIF) Figure S7 Amino acid sequence comparisons between CnaA-1, CnaA-2, and CnaA. The CNAA-1 allele encodes threonine (T) instead of serine (S) in cnaA at the 378 th residue; the CNAA-2 allele encodes aspartic acid (D) instead of asparagine (N) in cnaA at the 370 th residue. The amino acid alterations are present in the binding domains for calcineurin B and for the FKBP12-FK506 complex and, therefore, the interaction between FKBP12-FK506 and calcineurin may be modified to confer resistance to FK506.  (Table S1). (B) P1 and P4 primers amplified a 4332 bp region of the cnbRD::pyrG allele from two independent cnbR mutants, whereas the same primers amplified only a 2958 bp cnbR fragment from wild-type. P1 and P2 primers amplified 1658 bp from the 59 junction of the cnbRD::pyrG allele, and the P3 and P4 primers amplified 1895 bp from the 39 junction of the cnbRD::pyrG allele. The same pairs of primers did not produce junction fragments from the wild-type. The gene and primer sizes are not to scale. M1: MSL7 and M2: MSL8 (Table 1) Wax moth larvae were infected with Mucor (20,000 spores or yeast) and after two days, the larvae were frozen and sliced for microscopic observation. Fungal mass in tissues was stained with a 0.05% solution of calcofluor white. The wild-type displayed hyphal growth inside of infected tissues, whereas the two independent cnbRD mutants only grew as yeast inside of the wax moth larva hosts. Scale = 20 mm. CFW: calcofluor white, DIC: differential interference contrast. (B) Mice were infected with Mucor or Rhizopus, and the brains from infected mice were collected and tissue specimens were stained with Gomori's methenamine silver (GSM) at 2 days post infection. Both fungi displayed hyphal growth in the recovered brain tissues. Scale = 50 mm.  (Table S1). (B) P1 and P4 primers amplified a 4447 bp product for the cnaAD::pyrG allele from two independent cnaA mutants, whereas the same primers amplified only a 4791 bp cnaA fragment from wild-type. P1 and P2 primer amplified a 1582 bp region from the 59 junction of the cnaAD::pyrG allele, and the P3 and P4 primers a 2113 bp product from the 39 junction of the cnaAD::pyrG allele from the two cnaA mutants. The same pairs of primers did not produce junction fragments from wild-type. The gene and primer sizes are not to scale. M1: MSL9 and M2: MSL10 (Table 1). (TIF) Figure S13 Southern blot confirmation of the disruption of the cnaA gene. Genomic DNA (30 mg) of wild-type (MU402), MSL9 (cnaAD1), and MSL10 (cnaAD2) were fully digested with BamHI. The 39 UTR end of the cnaA gene was amplified and labeled with P 32 . The probe sequence is a part of the disruption cassette. The probe detected a 10,461 bp wild-type BamHI fragment, whereas a 5,442 bp BamHI fragment was detected in the cnaAD mutants, confirming a deletion of the cnaA gene in the cnaAD mutants. No apparent extra signals were detected, indicating that no ectopic integration events occurred. The gene sizes are not to scale. (TIF) Figure S14 Morphology of cnaA mutants in the wax moth larva host. Wild-type and two independent cnaAD mutants (20,000 spores) were inoculated into wax moth larvae. After two days, the larvae were frozen and sliced for microscopic observation. A drop of 0.05% calcofluor white solution was applied to stain fungal mass in the tissues. Both of the wild-type and cnaAD mutants formed hyphae inside of the wax moth larva hosts. Scale = 10 mm. (TIF) Figure S15 Larger spores of the cnaA mutants germinate earlier than wild-type. J774.A1 macrophage cells (5610 5 ) were inoculated into each well of a six-well plate. After 24 hours of incubation, 5610 5 spores were co-cultured with the macrophages. The interactions between macrophages and spores were observed every 30 minutes for 6 hours. The larger cnaA spores germinated earlier, at 3.5 hours, inside macrophages than the wild-type, at 6 hours. The images were taken at 3.5 hour. (TIF) Figure S16 Effect of FK506 on diabetic (upper) and nondiabetic (bottom) murine hosts infected with M. circinelloides or R. oryzae. Groups of BALB/c mice were rendered diabetic with streptozocin (190 mg per body kg) through intraperitoneal injection 10 days prior to fungal challenge [53,96]. The mice were infected with 10 4 Mucor (CNRMA04.805) spores or 10 3 Rhizopus (RA99-880) spores in 200 mL PBS via tail vein injection. The survival rate of the hosts was monitored twice a day, and body weight was measured daily. Animals that appeared moribund or in pain were sacrificed. The significance of mortality data was evaluated with Kaplan-Meier survival curves. The mice infected with Mucor or Rhizopus spores were immediately administered FK506 at a concentration of 5 mg per kg of body weight via intraperitoneal injection. Two additional FK506 treatments were performed at the same concentration at 24 and 48 hours postinfection. In both groups of mice infected with Mucor or Rhizopus, no apparent therapeutic effect of FK506 was observed. Nondiabetic murine host models were also tested, in which 10 5 Mucor spores and 10 4 Rhizopus spores were intravenously injected. FK506 at 5 mg per kg of body weight was administered at 0, 24, and 48 hours post-infection. No apparent therapeutic benefit of FK506 was observed in this analysis. A detrimental effect was observed in the mice infected with Mucor (p = 0.0015). It is possible that in both the diabetic and non-diabetic murine host models immunosuppression due to FK506 might have counteracted the antifungal effects of the drug. These results suggest that lower doses of FK506 (altered dosage regimens), less or non-immunosuppressive FK506 analogs, or a combination of FK506 with other antifungal drugs will be necessary for therapeutic efficacy. (TIF) Figure S17 One hundred kilobases upstream and downstream of the three cna genes are depicted. The 59 and 39 flanking regions of the three cna genes are not syntenic; however, repetitive elements are present. The blue-arrow repetitive sequences (,1 kb) are found in the flanking areas of all three cna genes; the greenarrow repetitive elements (,500 bp) are found in the flanking areas of the cnaA and cnaC genes; the red-arrow and gray-arrow repetitive elements are found only in the flanking area of the cnaA or cnaB gene, respectively. These observations illustrate that the three cna genes may have evolved from multiple segmental gene duplication events, which may have been facilitated by these or other repetitive elements. (TIF) Video S1 Hyphal growth of M. circinelloides. The wild-type R7B strain was grown on YDP solid media in aeration conditions. Mucor exhibited a filamentous growth forming mycelial complex.