Phenotypic characterization of HAM1, a novel mating regulator of the fungal pathogen Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans is a fungal pathogen responsible for >200,000 yearly cases with a mortality as high as 81%. This burden results, in part, from an incomplete understanding of its pathogenesis and ineffective antifungal treatments; hence, there is a pressing need to understand the biology and host interactions of this yeast to develop improved treatments. Protein palmitoylation is important for cryptococcal virulence, and we previously identified the substrates of its main palmitoyl transferase. One of them was encoded by the uncharacterized gene CNAG_02129. In the filamentous fungus Neurospora crassa, a homolog of this gene named hyphal anastomosis protein 13 plays a role in proper cellular communication and filament fusion. In Cryptococcus, cellular communication is essential during mating; therefore, we hypothesized that CNAG_02129, which we named hyphal anastomosis protein 1 (HAM1), may play a role in mating. We found that ham1Δ mutants produce more fusion products during mating, filament more robustly, and exhibit competitive fitness defects under mating and non-mating conditions. Additionally, we found several differences with the major virulence factor, the polysaccharide capsule, that may affect virulence, consistent with prior studies linking virulence to mating. We observed that ham1Δ mutants have decreased capsule attachment and transfer but exhibit higher amounts of exopolysaccharide shedding and biofilm production. Finally, HAM1 expression is significantly lower in mating media relative to non-mating conditions, consistent with it acting as a negative regulator of mating. Understanding the connection between mating and virulence in C. neoformans may open new avenues of investigation into ways to improve the treatment of this disease. IMPORTANCE Fungal mating is a vital part of the lifecycle of the pathogenic yeast Cryptococcus neoformans. More than just ensuring the propagation of the species, mating allows for sexual reproduction to occur and generates genetic diversity as well as infectious propagules that can invade mammalian hosts. Despite its importance in the biology of this pathogen, we still do not know all of the major players regulating the mating process and if they are involved or impact its pathogenesis. Here, we identified a novel negative regulator of mating that also affects certain cellular characteristics known to be important for virulence. This gene, which we call HAM1, is widely conserved across the cryptococcal family as well as in many pathogenic fungal species. This study will open new avenues of exploration regarding the function of uncharacterized but conserved genes in a variety of pathogenic fungal species and specifically in serotype A of C. neoformans.

the importance of morphological transitions for pathogenesis.Histoplasma capsulatum grows naturally as a mold in the environment, but under host conditions, it grows as a budding yeast, which allows for greater dissemination in the bloodstream (3).Conversely, Aspergillus fumigatus conidia are inhaled into the lungs and germinate into hyphal filaments, which can form mycelial networks that are able to invade into host tissues and, due to their large size, are unable to be phagocytosed by host immune cells.In the case of Cryptococcus neoformans, one such morphological transition happens prior to host invasion, where sexual and asexual reproduction produces the spores and desiccated yeasts that are the infectious agents inhaled into the lungs to establish infection (4).
C. neoformans is an opportunistic basidiomycete pathogenic fungus present worldwide (4).Because it is in the environment, the infectious propagules are frequently inhaled into the lungs, causing an infection that, in healthy individuals, is controlled and remains asymptomatic until cleared.However, in the immunocompromised, the infection quickly overwhelms the immune system in the lungs, resulting in pneumonia and acute respiratory distress syndrome, with subsequent dissemination to the central nervous system, causing lethal meningoencephalitis (5).C. neoformans is a leading cause of death in HIV/AIDS, where there are approximately 152,000 cases each year with 112,000 resulting in death (6).This staggering mortality rate stems largely from a lack of effective treatment options as well as an incomplete understanding of cryptococcal pathogenesis.This has prompted the World Health Organization to include C. neofor mans in the highest critical priority group of its recent Fungal Priority Pathogen List (7).
In a previous study investigating cryptococcal factors necessary for phagocyte recognition and internalization (8), we identified a gene homologous to Saccharomyces cerevisiae's protein fatty acyltransferase 4 (PFA4).We and others showed that cryptococ cal PFA4 was necessary for complete virulence in a mammalian host (8,9).Since palmitoyl acyl transferases (PATs), like Pfa4, act by modifying substrate proteins, we determined its palmitoylome.Using comparative mass spectrometry between wild-type (WT) and pfa4Δ samples to determine downstream targets, one of the most highly enriched substrates was encoded by the gene of unknown function, CNAG_02129 (8).
CNAG_02129 is part of a conserved family of fungal genes of which only one member, from the filamentous ascomycete Neurospora crassa, has been partially characterized (hyphal anastomosis protein 13 or HAM-13) (10) (Fig. 1A).In the absence of HAM-13, only 33% of the N. crassa germlings find each other and fuse.This is in stark contrast to almost 100% of the WT germlings finding each other, undergoing cellular fusion, and maturing into interconnected hyphae to form a mycelium (11).Thus, the authors concluded that HAM-13 plays a role in cellular communication.In C. neoformans, this type of cellular communication is essential for mating, which can be either unisexual or bisexual depending on the surrounding environmental pressures such as nutrient limitation or pheromone secretion (12).Members of the C. neoformans species complex (which includes seven pathogenic species) have two mating types determined by the mating-type (MAT) loci, MATα and MATa.In a bisexual scenario, MATα and MATa cells find each other through pheromone signaling, which triggers a cell fusion event resulting in a morphological transition into filamentous hyphae.The nucleus from each parent travels up these hyphal structures to the apex where a basidia is formed.Within the basidia, nuclear fusion occurs followed by meiosis and several rounds of mitosis to produce four chains of haploid basidiospores.These spores then drop from the basidium and mature into yeast where the process can be started again.In a unisexual scenario, the same events occur but only in cells containing the MATα locus (12).Almost all of these details, however, are from studies using C. neoformans serotype D (now called C. deneoformans), and considerably less is known about C. neoformans strains that are serotype A (C. neoformans sensu stricto).
Several studies have shown an inverse relationship between C. neoformans's virulence and its filamentous mating form (15,16).For example, when ZNF2, a master regula tor of filamentation in C. neoformans, is constitutively overexpressed, the cells are morphologically hyphal locked and unable to cause disease in a mouse model (17).These hyphal-locked cells induced a protective TH-1 pro-inflammatory immune response that not only resulted in full clearance of the initial infection but also protection from subsequent challenges (18).This suggests that mating regulators may also play a larger role in the control of virulence mechanisms, such as impacting any of the main cryptococcal virulence factors: its polysaccharide capsule, thermotolerance, and melanization.These and other factors allow for C. neoformans to persist within the harsh environment of a human host.While each of these virulence factors has been comprehensively studied, not all their regulators have been uncovered.
In this study, we determined that CNAG_02129, which we refer to as HAM1 (hyphal anastomosis protein 1), plays a role in cryptococcal mating by acting as a negative regulator of the process and has a connection to proper polysaccharide capsule retention and shedding.Our ham1Δ strains exhibit increased cellular fusion that was not augmented further by exogenous pheromone, a hyper hyphal response during bisexual mating scenarios, and reduced competitive fitness under both mating and non-mating conditions.Interestingly, these effects were specific for C. neoformans (serotype A) and not obvious in C. deneoformans (serotype D), highlighting the known differences in mating regulation between the sister species.Notably, although HAM1 expression is significantly downregulated during mating conditions, there are no differences in MAT loci pheromone expression in the ham1Δ, suggesting a fork somewhere in the pheromone signaling cascade.Consistently, HAM1 expression still is high under mating conditions in the mat2Δ mutant but not in a cpk1Δ strain, both of which are sterile mutants with no pheromone expression.Although we do not observe major defects in capsule generation and other key virulence mechanisms, we found defects in capsule attachment, an increase in capsule shedding, less effective capsule transfer, and an increase in biofilm production.These findings suggest a connection between mating and proper capsule formation and shedding, which can impact the virulence potential of this organism and contribute to our understanding of mating regulation in this important fungal pathogen.

Ham1 is a palmitoylated protein conserved across the fungal kingdom
To confirm the results of our previous large-scale Pfa4 palmitoylome studies, we investigated the palmitoylation status of Ham1 using a click chemistry-based palmitoyla tion probe (8).Using this probe (alk15; an analog of palmitate) and a HAM1-FLAG tagged strain (see Materials and Methods section), we performed an immunoprecipitation (IP) coupled with click chemistry to determine if the Ham1 protein was palmitoylated.We confirmed that Ham1 is palmitoylated by the presence of a fluorescent band only in the probe-fed fraction of our Ham1-FLAG IP that was at the same size as the accompanying western blot (Fig. 1B).Next, we wanted to see if the palmitoylation modification may be conserved across the protein homologs.Using the palmitoylation predictor software GPS-Palm (14), we determined that all of the homologs investigated had very high confidence predictions for palmitoylation (Fig. 1C).

ham1Δ exhibits altered cellular fusion and progeny with a dry colony morphology
The goal of mating is to produce diploids that can undergo meiosis and generate haploid cells with traits from both parents, generating better-adapted cells (12).This requires that parental strains find each other, fuse, undergo recombination, and then produce haploid spores.To test if mating was affected, deletion strains of HAM1 were generated in both mating types of the KN99 background (WT) using different dominant markers (NEO or NAT).We then set up unilateral (one mating partner is mutant, the other is WT) and bilateral (both mating partners are mutant) mating crosses between WT mating controls (KN99-NEO or KN99-NAT) and ham1Δ mutants (ham1Δ-NAT) and determined the fusion efficiency from the number of colonies resistant to both antibiotics.Both unilateral and bilateral ham1Δ mutant crosses yielded significantly higher colonies resistant to both antibiotics when compared to the WT cross, indicating faster, or more efficient, cellular fusion (Fig. 2A; Fig. S1).Notably, the ham1Δ mutants are still under pheromone regulatory control, as there are no fusion events detected between the same mating types (Fig. 2A).However, to explore the impact that pheromone might have on cellular fusion, we tested if adding synthetic exogenous pheromone would increase the rate of cell fusion in our ham1Δ mutants.Although addition of exogenous pheromone to the WT cross resulted in an increase in the number of colonies generated, there was no significant difference in the fusion rate (Fig. S1A).Likewise, in our ham1Δ crosses, there was no statistical significance by one-way analysis of variance (ANOVA) with multiple comparisons (Fig. S1B through D), indicating that this enhanced fusion is pheromone signal independent.Interestingly, when scoring double-resistant colonies, we observed a difference in their morphology.Rather than the typical glossy, round-shape colonies coming from the WT strain crosses, the fusion products from the ham1Δ crosses had a dry, irregular morphology (Fig. 2B).When resuspending single colonies from these crosses and staining them with 4′,6-diamidino-2-phenylindole (DAPI) and Calcofluor white (CFW) to visualize, we found that these colonies were comprised of the hyphal form of Cryptococcus, as the septal divisions and a bi-nuclear distribution along septa were evident (Fig. 2B).In contrast, the colonies from the WT crosses only showed budding yeasts.This drastic increase in the ability to fuse as well as the dry appearance and hyphal state of ham1Δ progeny led us to consider what other facets of the mating cycle could be altered.

ham1Δ mutants have increased hyphal production in bisexual mating scenarios on V8 medium
Hyphal growth is a quantitative trait that can be used to measure how well a strain is able to mate (19).We assessed the ability of our ham1Δ crosses to form hyphae in a time course from 5 days to 4 weeks.We observed faster and more robust filamentation in our ham1Δ crosses across all time points (Fig. 3A).When quantifying the hyphal area (see Materials and Methods section), we observed a significant increase in all unilateral mutant crosses at 5 days and 3 weeks and a significant increase in the hyphal area of the bilateral crosses across all time points (Fig. 3B).This was confirmed using two additional independent ham1Δ mutants, including one from the deletion collection available commercially (Fungal Genetics Stock Center; Fig. S2A).

ham1Δα strain exhibits a fitness disadvantage in both mating and non-mat ing scenarios
Mating is an energy-costly process and may result in a fitness cost when comparing mating and non-mating conditions.However, this is crucial for survival in the environ ment where mating partners are few and far between, and the pressures of predators and other stressors threaten survival.We tested if our ham1Δ would display increased fitness compared to a WT competitor due to its propensity to form more hyphae and increased cellular fusion events.To test this, we had two MATα or two MATa strains with opposing resistance cassettes (the competitors) co-incubated with the opposing mating type with no resistance cassette (the donor) (Fig. 4A).All three strains were mixed and plated under mating (V8) or non-mating [yeast extract peptone dextrose (YPD)] conditions for 10 days.At that point, each competitive scenario was scraped, resuspended, and plated on selective media to determine the colony-forming units (CFUs) of each competitor and calculate a competitive index (CI; Fig. 4B).In the mating permissive condition (V8), we expect both competitors to stop vegetative budding and forage for mating partners, while in the non-mating condition (YPD), we expect both competitors to simply divide.In the WT cross, we observed no fitness defects (CI ≈ 1) in either YPD or V8 media conditions (Fig. 4C).However, in our ham1Δ mutant crosses, we saw CI values significantly different from 1.In the ham1Δα competition, the CI was less than one (0.17 in YPD and 0.32 in V8), indicating significant fitness defects for ham1Δα in both the V8 and YPD conditions (Fig. 4C).Surprisingly, in our ham1Δa competition, we observed the opposite: CI values larger than one (1.84 in YPD and 1.65 in V8) showing a strong competitive advantage in both conditions for ham1Δa (Fig. 4C).

Transcriptional analysis of the pheromone pathway and HAM1 over time
To determine if HAM1 is involved in the pheromone response mitogen-activated protein (MAP) kinase pathway, we analyzed the transcriptional activity of the MATα/MATa pheromone loci and HAM1 under YPD and V8.This type of analysis is typically done in C. deneoformans, but when we deleted HAM1 in this species, mating was unaffected (data not shown).Since it is known that there are differences in mating regulation between the two sister species, we decided to continue and analyze the transcription of these genes in C. neoformans.To do this for the pheromone loci, we compared the gene expression of MATα and MATa in mating conditions normalized to the expression in non-mating conditions to get the relative fold induction (20,21).We performed this in a time course of 1, 3, 5, and 7 days to look at changes in expression over time (Fig. 5A through C).Not surprisingly, we observed no statistically significant differences in either MATα or MATa expression between the crosses.However, in the WT cross, we saw a strong and consistent downregulation of HAM1 under mating conditions relative to non-mating media.We saw that on day 1 in mating conditions, HAM1 had a much lower expression compared to non-mating conditions (Fig. 5C).However, as the mating cycle progressed, the expression of HAM1 returned to levels similar to those under non-mating conditions (Fig. 5C).These findings, taken together with the increased fusion rate and filamentation of the ham1Δ mutants, suggest that HAM1 acts as a negative regulator of mating (inhibiting mating under non-mating conditions).This would imply that the pheromone MAP kinase pathway downregulates expression of HAM1 to promote mating.To test this, we measured HAM1 expression in WT, cpk1Δ, and mat2Δ mutants under both mating and non-mating conditions (Fig. 5D).Consistently, in bisexual crosses between either of these mutants [known to be sterile mutants (22)] and KN99a, HAM1 expression remains high.The same happens when only one mating partner is grown in YPD or V8 (both would be non-mating conditions for C. neoformans, as this species does not undergo unisexual reproduction): the relative HAM1 expression is even higher in V8 than in YPD.Interestingly, when measuring HAM1 expression in monocultures of cpk1Δ or mat2Δ, the mat2Δ cells still greatly induce HAM1 in V8 relative to YPD just like the WT monocultures, whereas in the cpk1Δ monoculture, HAM1 was downregulated similar to WT bisexual crosses.

ham1Δ has no significant defects in major virulence mechanisms
So far, all the phenotypes of the ham1Δ have been mating related, especially in the MATα background.We wondered if these would also affect virulence; hence, we investigated if there were defects in the main virulence factors of C. neoformans: capsule production, melanization, thermotolerance (37°C), and recognition by phagocytic cells.We found no obvious differences in any of the major virulence factors (Fig. 6; Fig. S3, and data not shown).Due to the surface defects observed in the pfa4Δ mutant (Ham1's PAT) (8) we took a closer look at the cell wall and the capsule of our ham1Δ strains as more nuanced differences could be present besides capsule induction.Finding no apparent defects under different cell wall and membrane stressors (Fig. S3), we took a closer look at the polysaccharide capsule and investigated differences in its attachment and release.

ham1Δα exhibits defects in capsule attachment and transfer
First, we looked at capsule attachment.After inducing capsule formation, we subjected WT, ham1Δα, and a known capsule mutant (pbx1Δ) to sonication (Fig. 7A).We found that our ham1Δα mutant lost significantly more capsule than WT cells, suggesting a defective attachment to the cell wall (Fig. 7B; Fig. S2).The next avenue we investigated was capsule transfer.We incubated the acapsular mutant cap59Δ with conditioned media from WT, ham1Δα, pbx1Δ, and cap59Δ to see how well the capsular material could attach to the cell wall of cap59Δ (Fig. 7C).As expected, our cap59Δ mutant could not transfer a capsule at any dilution tested due to the lack of a capsule.Our pbx1Δ mutant also was unable to transfer a capsule at dilutions higher than 1:5, which was unsurprising due to its known capsule defects (23,24).However, our ham1Δα also struggled to transfer the capsule at higher dilutions > 1:750, whereas the WT capsule had no problem at these dilutions (Fig. 7C; Fig. S2).This indicates at least a small defect in either structure or reduced release of capsule.Defects indicated by sonication and transfer in our ham1Δα prompted us to look at shedding and biofilm production, which could further confirm issues with capsular release (25).

ham1Δα has altered capsule shedding in different media and higher biofilm production
To investigate the possibility of defects with capsule release, we assessed the ability of ham1Δα to shed capsule in different media by electrophoresis and immunoblotting (25, 26) (Fig. 8A).We observed specific differences in the shed polymer size in nutrient-rich YPD medium with potential differences in size and amount in the capsule-inducing medium Dulbecco's modified Eagle's medium (DMEM) and the minimal growth medium yeast nitrogen base (YNB) (Fig. 8A).To get a more quantitative measurement of amounts sheds, we measured glucuronoxylomannan (GXM) concentration by enzyme-linked immunosorbent assay (ELISA) (25).In YPD, we saw a significant increase in the amount of capsule shed in our ham1Δα (Fig. 8B; Fig. S2).We saw no significant difference in the amount of capsule material shed in YNB or DMEM relative to WT (Fig. 8B through D; Fig. S2).For all media, our capsule-shedding mutant pbx1Δ shed less than WT, as expected, and our acapsular mutant cap59Δ did not shed any capsule.Given the association between capsule and biofilm formation, we next assessed how well ham1Δα could produce a biofilm using the 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5carboxanilide (XTT) biofilm assay (25).We found that our ham1Δα produced larger biofilms than our WT as did pbx1Δ, whereas no biofilm was produced in the acapsular mutant cap59Δ (Fig. 8E).

ham1Δα shows no difference in virulence capacity in G. mellonella
Finally, to investigate whether the role of HAM1 in mating and proper capsule function translates to virulence defects, we performed infections using the invertebrate model system Galleria mellonella (27).We infected G. mellonella with 1 × 10 5 log phase cells/ larvae and, surprisingly, saw no significant difference in survival between WT and ham1Δα-infected larvae (Fig. 9A; Fig. S2).On the other hand, the pbx1Δ mutant showed attenuated virulence, which is consistent with previous studies (23).We also assessed fungal burden at time of death, but saw no significant difference in the fungal burden of any of the strains (Fig. 9B; Fig. S2).

DISCUSSION
Mating is a vital part of the lifecycle of the C. neoformans species complex.It allows for sexual reproduction, increasing the genetic diversity among progeny and overall propagation of the species.Despite having a well-defined sexual cycle both in unisexual and bisexual scenarios, not all the important regulators of mating are known, especially in C. neoformans (serotype A strains).Our results presented here are consistent with HAM1 acting as a negative regulator of mating specifically in C. neoformans, with additional direct or indirect roles pertaining to the capsule.Moreover, the mating-associ ated morphological changes have been suggested to impact infection in multiple ways (C) Representative images of capsule transfer assay; capsule visualized with Alexa488-conjugated 3C2 antibody and cell wall with CFW.CM, conditioned media.Scale bar is 10 µm.(15,16); hence, understanding how it is regulated may yield novel and more effective ways to prevent, or treat, this disease.Given the proposed role of HAM-13 in N. crassa, we focused on the ability of cryptococcal ham1Δ mutants to fuse and produce recombinant progeny.Throughout our investigations, ham1Δ mutants consistently outperformed the WT strain in hyphal formation and production of progeny.This suggests that HAM1, like its homolog in N. crassa, is somehow connected to cell-to-cell communication and fusion of fungal cells.However, these phenotypes were only present in C. neoformans, and not in C. deneoformans, precluding us from interrogating more in-depth the potential signaling pathways affected by HAM1.Mating studies in Cryptococcus have been mostly done in serotype D strains (C.deneoformans) rather than in serotype A, as in this study, because of the natural propensity for mating in C. deneoformans.Hence, although surprising, it is not unexpected that HAM1 deletion yields different phenotypes between the sister species.Additionally, we found that the HAM1 effects in C. neoformans were exclusively found in the V8 mating-inducing medium as there was no significant increase in the amount of filamentation observed in either Murashige-Skoog or filamentation agar (data not shown).V8 media is considered the "gold standard" for looking at mating interactions in C. neoformans, while the others are mostly used in C. deneoformans (28), potentially explaining why we only see phenotypes in V8.Given that mating in C. neoformans is less well understood, and considering the larger epidemiological impact of serotype A strains, which comprise 95% of clinical isolates and have been associated with higher virulence potential (29,30), we chose to retain the serotype A strain for these mating assessments.Still, as discussed below, prior transcriptomic data published for C. deneoformans show that HAM1 levels are dramatically downregulated under mating conditions (31), relative to YPD, similar to our findings in C. neoformans.
The potential role of HAM1 in filamentation was very apparent in our ham1Δ cellular fusion assay, where colonies exhibited a dry desiccated form, which were, in fact, aggregates of hyphae.However, when these dry colonies were re-streaked, they returned to a "normal" colony morphology.The presence of the filamentous form in the absence of any mating pressure shows that the progeny of our ham1Δ strains have an even stronger drive toward the mating form but that, over time, the signaling to return to a normal yeast state is re-established.This, however, could impact the fitness of these strains under non-mating conditions.Thus, we were surprised to find that our ham1Δα has a fitness disadvantage in both mating permissive and non-mating conditions, whereas our ham1Δa performed better in mating permissive and non-mating conditions.One of the main differences between cellular competition and other classical mating assays in C. neoformans is that in the latter, opposite mating-type partners are in very close proximity, which masks issues with searching for mating partners.In our ham1Δα competitions under mating conditions, ham1Δα, while good at making hyphae and producing progeny with a partner in close proximity, may lack the ability to effectively forage for mating partners in nonstatic conditions.The disadvantage in non-mating conditions may be the result of two possibilities.First, ham1Δα has a drive to mate that outweighs the drive to cellularly divide in the presence of an opposite mating partner.Second, in S. cerevisiae, prolonged exposure to mating pheromones results in program med cell death (32).This same principle may apply in this case in that the presence of an opposite mating partner and the generation of MATα pheromone may induce inhibition of cellular growth or death in ham1Δα cells.Surprisingly, in our ham1Δa competition, we observed an increase in competitive fitness in both non-mating and mating conditions.These observations may be the result of the differences in the morphological changes happening to the different mating types.For example, in C. deneoformans, the MATα cell responds to pheromones by forming conjugation tubes, while the MATa cells become enlarged (33).To the best of our knowledge, these pre-cellular fusion structures have been observed only in C. deneoformans and are defined by their MAT loci (34)(35)(36).If this pre-cellular fusion morphology occurs in C. neoformans, it is possible that in the mating permissive competitive scenario with the ham1Δa strain, the WT MATα mating partner is able to produce conjugation tubes and effectively seek the MATa mutant partner.This would not happen with the ham1Δα strain seeking the WT MATa partner, explaining the different results.This also highlights the possibility that HAM1 may have a greater effect on MATα strains relative to MATa ones.This would not be a new concept.A previous study looking at the global transcriptional repressor TUP1 found mating-type specific phenotypes (37).Disruption of TUP1 led to different gene expression profiles in MATα vs MATa despite having very similar cellular fusion and filamentation phenotypes.This finding implies that even among lab strains, there is heterogeneity with respect to competitive fitness depending on the MAT locus and that not all WT strains will behave the same when in a competitive environment.
We wondered if HAM1 was acting on the pheromone MAP kinase pathway, but we were unable to obtain clear results from the qRT-PCR.This was not surprising given that serotype A strains do not respond to mating signaling uniformly or at the same time; hence, the RNA samples represent a mixture of states where mating cells are a minority.This was the case when measuring MATα, MATa, PUM1, and CFL1 during unilateral and bilateral crosses, where we saw no statistically significant differences relative to WT crosses.Still, we saw a consistent and significant repression of HAM1 on V8 early on, as expected if acting as a negative regulator of mating.Notably, repression of HAM1 would occur in V8 only in the presence of both mating partners, since in monocultures of MATα or MATa strains, HAM1 was induced in V8 relative to YPD, again consistent with it being a negative regulator of mating.Interestingly, in mat2Δ monocultures in V8, we still saw an induction of HAM1, whereas in cpk1Δ monocultures, missing Mat2's upstream kinase, HAM1 was repressed.This is consistent with a model where upon pheromone pathway activation, HAM1 is repressed in a Cpk1-dependent but Mat2-independent manner.However, in the absence of pheromone activation, HAM1 is induced, indicating additional regulatory inputs into HAM1.While mating is predominantly controlled by the pheromone response pathway, previous studies have shown that calcineurin signaling is essential for mating, haploid fruiting, and spore production (38,39).Hence, we tested a potential involvement of HAM1 in that pathway by assessing sensitivity to calcium stress, but found no defects with our ham1Δ mutants (Fig. S3).Still, this does not rule out the possibility that calcineurin impinges on HAM1 regulation.In fact, a study looking at the calcineurin-regulated genes necessary for thermotolerance found HAM1.Upon shift from 24°C to 37°C, there is a 14-to 17-fold increase in HAM1 levels, while in the cna1Δ, it is only 8-fold (39).
One of the hallmarks of mating in C. neoformans is the morphological transition from yeast to hyphae.The hyphal form of C. neoformans has been shown to be unable to cause disease in a mammalian host, in part, because it induces an immune response shift from a disease-permissive TH-2 to a disease-protective TH-1 proinflammatory response.When examining previously published RNA-seq data, we noted that HAM1 transcripts were some of the highest induced in cryptococcosis patients (40,41).This finding is intriguing for two reasons.First, it lends support to the idea that to prevent a morpholog ical transition in this low nutrient environment, negative regulators of mating would be highly expressed.Second, it may indicate additional roles for HAM1 directly related to maintenance of virulence factors providing further evidence of the link between mating and virulence.However, our initial survey of the key virulence mechanisms did not show any obvious defects in ham1Δ mutants.Still, we decided to take a closer look at the capsule since, as a downstream target of Pfa4, it is possible that ham1Δ may share defects related to the cell wall or capsule as exhibited by the pfa4Δ mutant.Additionally, since one of the major phenotypes in the ham1Δ relates to filamentation, we wanted to investigate if there was a possible crossover between the cellular circuits governing morphology and capsule production and maintenance.
Although capsule induction was similar, ham1Δ mutants exhibited a defect in capsule attachment and shedding.This was similar to phenotypes exhibited by pbx1Δ and pbx2Δ strains, both mutants with abnormal capsule structure (23,24).While defects were milder than pbx1Δ, capsule attachment and transfer were reduced in the ham1Δ.Capsular shedding, however, was increased in YPD, a capsule-non-inducing medium, and trended toward higher shedding in capsule-inducing media.This suggested a level of involvement of HAM1 in proper capsule formation that could be a direct or an indirect consequence of dysregulated mating signaling.Capsule regulation and structure during mating conditions is a topic that has received little attention.It is reasonable to envision changes in capsule accompanying the morphological changes present during mating.Hence, these capsule defects seen in ham1Δ mutants might be a consequence of the abnormal mating signaling occurring in the absence of HAM1.Consistently, normal capsule production is required for biofilm formation.Biofilm production, while not seen as a virulence factor for C. neoformans, was likely developed as a survival tactic against environmental predation (42).The ham1Δα mutants have altered biofilm formation, again suggesting defects in capsule attachment and altered capsule structure.With all the similarities in capsule defects shared between pbx1Δ and ham1Δα, we next wanted to assess the virulence capabilities of ham1Δ.
Deletion of pbx1Δ results in attenuated virulence in a mouse model, so we expected to see a similar defect in our ham1Δα (23).To our surprise, there were no significant defects in virulence potential using the G. infection model relative to WT (median death rate: WT, 4 days; ham1Δα, 5 days; and pbx1Δ, 6 days).Likewise, there were no differences in fungal burden, even in the pbx1Δ-infected larvae.This could be attributed to the fact that they lived longer, as the burden was assessed at the time of death.More detailed virulence assays will be needed to confirm if there are defects in ham1Δ mutants affecting its pathogenesis.
Taking all of these findings together, we propose a model where HAM1 negatively regulates the mating cycle of C. neoformans (Fig. 10).We envision two possible locations where it could be acting: first, it could act directly on pheromone-activated genes, inhibiting their function; or second, it could be directly involved in the yeast to hyphal transition.Although we do not have evidence for it, it is possible that it is acting at later stages as an inhibitor of recombination or spore generation, although it is unlikely given that we see HAM1 expression changes only at day 1 under mating conditions.Regardless, by acting as a negative regulator of the mating cycle, we believe that it prevents mating interactions from occurring during pathogenic conditions.This could be a potential reason HAM1 is so highly expressed in the CSF of cryptococcosis patients.CSF is a very nutrient-limited environment, which may provide the right pressures for cells to undergo a mating interaction, but that would be detrimental to pathogenesis.The signal to activate HAM1 under non-mating conditions, however, remains unclear (Fig. 10).
By uncovering a role for HAM1 in C. neoformans biology, we have shown another potential intersection point between virulence and mating.Both processes are vital to the propagation and survival of the species, and the presence of regulators with Based on this study, we believe that HAM1 acts as a negative regulator in the mating cycle of C. neoformans.Upon activation of the pheromone-response pathway, HAM1 is repressed in a Cpk1-dependent manner.However, under non-mating conditions, HAM1 is induced by unknown signals.When induced, HAM1 regulates mating by inhibiting pheromone and pheromone-response genes directly, or somehow inhibiting hyphal formation directly.During pathogenic conditions, we believe that HAM1 is highly expressed to prevent mating from occurring due to the low-nutrient environment of a mammalian host.The changes we see in the capsule could be a direct effect of HAM1 or an indirect result of altered mating signaling in the absence of HAM1 (dashed lines).This figure was created with BioRender.com.roles in multiple pathways reflects just how important these are.Investigation of mating regulators not only improves our knowledge of how the system works but also opens new avenues of investigation for therapeutic interventions.Through a better understanding of fungal mating, we may be able to find more intersection points between virulence and mating pathways to fully understand how these two processes are connected.We hope this study serves to raise awareness about the importance of mating for virulence and stirs more investigations into this phenomenon.

Strains and growth materials
All serotype A strains were in the KN99 background (Table S1).All ham1Δ strains and HAM1-FLAG-tagged strain were generated using biolistic delivery (see methods below and Table S1).V8 agar medium was prepared using original recipe V8 juice, bacteriologi cal-grade agar, and distilled water (dH 2 O).V8 medium was adjusted to the appropriate pH (five for serotype A, seven for serotype D) using 1 M of hydrochloric acid or sodium hydroxide tablets, respectively.CuSO 4 (25 mM) was added after autoclaving before pouring into plates.

Phenotyping
Strains were grown overnight in 5 mL of YPD liquid culture diluted to an optical density measured at 600-nm wavelength (OD 600 ) of 0.2 and left to grow for two doublings.Strains were washed in 1× phosphate-buffered saline (PBS), counted using a Bio-Rad TC20 automatic cell counter, and diluted to a final concentration of 1 × 10 7 cells/mL.Cells were then serially diluted 10-fold to a final concentration of 1 × 10 3 cells/mL.Five microliters of culture was spotted onto the following plates: YPD, YNB, YPD with 1 M of NaCl, 0.5 mg/mL of caffeine, 0.1%-0.3%SDS, 0.35-0.5 M of CaCl 2 , 0.5 mg/mL of CFW oxidative (H 2 O 2 ) and nitrosative stress (NaNO 2 ).Plates supplemented with stressors were incubated at 30°C for 2-3 days.Plates that had no stressors were incubated at 30°C and 37°C to assess temperature defects.All plates were imaged on the Bio-Rad EZ Gel Doc Imager.

Fungal genomic manipulation
We generated all ham1Δ mutants using the split marker method to delete the entire HAM1-coding region.Fragments were delivered to candidate KN99α and KN99a cultures via biolistic delivery (Bio-Rad PDS-1000).Deletion candidates (from at least two independent transformations) were assessed using PCR as well as mating phenotype analysis to confirm deletions.Two of these independent deletion mutants were tested in all mating assays.To perform immunoprecipitations needed for the click chemistry analyses, a FLAG-tag strain was generated using the 4× FLAG-Tag plasmid to tag the C-terminal end of Ham1.This fragment with overlapping homology to the HAM1-coding sequence was synthesized by Genewiz and again delivered to candidate cells via biolistic delivery.This HAM1-FLAG-tagged strain was functional as it was indistinguishable from WT in mating and capsule assays.
In addition to generating deletion mutants via biolistics, we generated two independ ent ham1Δa candidates via mating of KN99a with ham1Δα (Table S1).Strains were grown in 5 mL of YPD for 48 h, spun down and washed twice in dH 2 O, counted, and normalized to a concentration of 1.5 × 10 7 cells/mL.Equal cell volumes of each mating pairs were mixed and incubated at room temperature (RT) for an hour.Of this mixture, 100-200 μL was spotted onto V8 mating plates and incubated in the dark at RT for 7 days.Filaments from each mating interaction were scraped using a sterile toothpick and resuspended in dH 2 O. Spores were plated on YPD with selection agar plates and streaked twice with selection for single colonies.Isolated single colonies were tested for mating type using a mating interaction with known tester strains (JEC20 and JEC21).These ham1Δa mutants behaved similar to the mutants generated by biolistics.We also generated a ham1Δα with the NEO cassette by clustered regularly interspaced short palindromic repeats (CRISPR)(Table S1) using the method published by the Madhani lab (43).
Finally, because we were unable to generate a complemented strain on the same loci using biolistics, we tested a commercially available ham1Δ deletion, which we refer to as C07 (the well number on the deletion collection plate).All three independent mutants showed the same phenotypes (Fig. S2 and data not shown).We also used a plasmid complementation approach by cloning the HAM1 locus (including its native promoter and terminator) into plasmid pMSC042-Neo (Fig. S4A) and electroporating into ham1Δα-NAT.We used this strain to asses complementation of the capsule attachment phenotype (Fig. S4B) and the biofilm formation alteration (Fig. S4C).

Cell preparation, protein extraction, and immunoprecipitation for clickchemistry
Using "clickable" fatty acid probes, such as alkynyl palmitate (alk15; Click Chemistry Tools #1165), which are metabolically incorporated into normal substrates by the cell, we can then carry out a bioorthogonal Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction (the click reaction) to conjugate the fatty acid-modified proteins to easily measurable reporters such as fluorophores or affinity tags.To confirm that Ham1 is palmitoylated, HAM1-FLAG and KN99a strains were grown in 50 mL of YNB medium, pH 7.0, and once an OD of 0.5 was reached, strains were fed with 15 µL of 5 µM alk15 and incubated for 1 h at 30°C with shaking.Cells were washed twice in cold dH 2 O and flash frozen until ready for use.Cells were thawed on ice and disrupted in lysis buffer (100 mM Tris-HCl pH 7.4, 0.5% Triton X-100, 150 mM of NaCl, 13% wt/vol glycerol, protease inhibitor tablet, Roche) by bead beating (mini bead beater-16, Biospec product #607) for 5 cycles of 1-min on, 1-min rest on ice.Cell extracts were spun down at 13,000 × g for 10 min at 4°C, and protein concentration in the supernatant was determined using a detergent-compatible Bradford assay (Thermo Scientific #23246).
Based on described methods (44), 1 µg of protein lysates from fed and unfed cell populations were incubated with 100 µL of 50% slurry of anti-FLAG M2 affinity gel (A2220; Sigma-Aldrich) for 20 min on a nutator at 4°C.The protein-bead mixtures were then loaded onto a gravity column (TALON 2 mL Disposable Gravity Column, Takara #635606).Lysate was loaded into the column five times with gravity flow and then washed in a 1 M of NaCl buffer three times.Beads were then washed twice with 150 mM of NaCl buffer before resuspended in Click Chemistry buffer according to the manufac turer's protocol (#1262, Click Chemistry Tools) apart from the AFDye 680 Azide Plus reporter (Click Chemistry Tools #1512), which was added at a concentration of 1.25 μM.Excess reactants were removed by spinning down anti-FLAG M2 affinity gel and washing twice in 1× PBS.Beads were then resuspended in 1× Laemmli Buffer and heated at 70°C for 10 min.After SDS-PAGE, the 10% polyacrylamide gel was destained in Click Chemistry Destain (40% methanol, 50% acetic acid, 10% water) overnight and gel visualized on an Odyssey Imaging System (LI-COR Biosciences).

Cellular fusion
Cellular fusion efficiency was assessed using overnight cultures grown in YPD that were washed twice in dH 2 O. Cultures were normalized to an OD of 0.5 and resuspended in dH 2 O.Each cross was comprised of cells of opposite mating type and different antibiotic resistance cassettes to determine the fusion efficiency of each cross.Equal volumes of each strain were mixed for their respective crosses and spotted in 50-µL increments on V8 mating agar.Each cross was spotted in triplicate and left to dry for 20 min.Once all plates were dry, they were transferred to a mating chamber (DM1, Mycolabs) and left in the dark for 5 days at ambient room temperature with a relative humidity of 40%-50%.After incubation, each cross was scraped and resuspended in 1 mL of dH 2 O and normalized via cell count to 1 × 10 7 cells/mL.For quantification, 100 µL of undiluted or 10-and 100-fold dilutions of the 1 × 10 7 -cells/mL suspension were plated on both double and single antibiotic YPD resistance plates (NAT and NEO).Once plates were dry, they were transferred to a 30°C room, incubated for 3 to 4 days, and counted for CFUs.

Hyphal staining
Single colonies from mutant crosses were scraped and resuspended in dH 2 O. Colonies were then stained with either 5 µg/mL of DAPI or 100 µg/mL of CFW and incubated at 30°C in the dark for 20 min.Cells were then washed three times in 1× PBS and resuspended in 100 µL.Cells were then imaged using a ×100 oil immersion objective in a Zeiss Axio Observer microscope.

Assessment of bisexual mating
This was adapted from the bisexual mating protocol from Sun et al., 2019 (28).Briefly, strains were grown overnight in YPD, washed twice in dH 2 O, and normalized to an OD of 1.Cells of opposite mating type for each cross were mixed in equal amounts in a separate tube and subsequently spotted in 20-µL amounts onto V8 agar plates.All crosses were spotted in triplicate and left to dry for 20 min.Once all plates were dry, they were transferred to a mating chamber (DM1, Mycolabs) and left in the dark for up to 4 weeks with a relative humidity of 40%-50%.

Cellular competition assessment
Each cross consists of three strains: two of the same mating type but with opposing antibiotic resistance cassettes (competitor strains), and one of the opposite mating type with no antibiotic resistance (donor strain).Overnight cultures for each strain were grown in YPD until log phase.Cultures were then washed twice in dH 2 O, counted, and normalized to a concentration of 1 × 10 5 cells/mL.Competitor and recipient strains were mixed in equal amounts for each cross.Of each cross, 30 µL was plated in triplicate on V8 and YPD medium and left to dry for 20 min.Once all plates were dry, they were transferred to a mating chamber and left in the dark for 10 days with a relative humidity of 40%-50%.After the 10-day crosses were scraped and resuspended in dH 2 O. Crosses in YPD were diluted 100,000×, and crosses in V8 were diluted 10,000×.Of the respective dilutions, 100 µL was plated in single antibiotic-supplemented YPD plates (NAT or G418).Plates were incubated for 2-3 days and imaged for CFU counting using a Bio-Rad Gel Doc EZ imager.

RNA isolation from bisexual mating crosses
Bisexual mating crosses were set up as previously described and allowed to incubate for 1, 3, 5, and 7 days.Cells were then harvested, washed once in dH 2 O, washed with RNA stop solution, and flash frozen and stored at −80°C until ready for extraction.A TRIzol-extraction (Life Technologies #15596-026) protocol is used followed by an RNA clean up step using the ZYMO RNA Clean and Concentrator kit (cat # R1019).Up to 1 ug of RNA was then converted into cDNA using the NEB LunaScript RT Supermix Kit (NEB #E3010) followed by qPCR using the NEB Luna Universal qPCR Master Mix (NEB #M3003).The ΔΔ CT method was used for quantification of relative transcript level normalized to YPD samples (45).

Capsule induction and visualization with India ink
Cultures were grown overnight in 5 mL of YPD medium.Cultures were spun down at 3,000 × g for 5 min and resuspended in 1 mL of PBS.After cell count, 1 × 10 7 cells/mL were transferred to a new microtube and pelleted.Pellets were resuspended in 1 mL of DMEM (Corning; VWR 45000-304).This suspension was diluted 10-fold by adding 500 µL to 4.5 mL of DMEM to a final concentration of 1 × 10 6 cells/mL.Of this cell suspension, 1-mL aliquots were added into a 24-well culture plate and incubated at 37°C with 5% CO 2 for 24 h.After 24 h of incubation, samples were transferred to microtubes and spun down at 3,000 × g for 5 min.Samples were washed in PBS and resuspended in a final volume of 50 µL.In a separate tube, 10 µL of sample was mixed with India ink, and a drop of the mixed sample and India ink was pipetted onto a polylysine-coated slide and imaged on a Zeiss Axio Observer Microscope.

Capsule sonication
The capsules were induced as described above, and the cells were resuspended in 1 mL of 1× PBS.A portion of the cells was aliquoted (unsonicated controls), and the rest was subjected to sonication using a tip sonicator (Branson #SFX250) at 20% power with 0.5 pulse for 20 s and kept on ice.Once all strains had been sonicated, all samples were mixed with India ink as described above and visualized on a Zeiss Axio Observer Microscope at ×100 oil immersion magnification.The capsule radius was measured using FIJI (46).

Capsule transfer
Capsule transfer assays were performed as previously described (24).Capsule donor strains were grown in 5 mL of YPD liquid culture for 5 days prior to the experiment.On day 4, a cap59Δ acceptor culture was grown overnight.Donor cultures were normalized to cell counts and spun down for 5 min at 3,000 × g, and the top 1.5 mL was filtered through a 0.22µm filter (Avantor #76479-024).From this filtration step, 1 mL was heated at 70°C to create conditioned media.Acceptor cells were washed twice with 1× PBS, counted on a Bio-Rad TC20 automatic cell counter, and diluted to a final concentration of 5 × 10 6 cells/mL in a volume of 400 µL.Conditioned medium was added in the appropriate dilution to acceptor cells and incubated for 1 h with gentle rotation.Cells were pelleted at 3,000 × g for 5 min and washed twice with 1× PBS.Cells were then resuspended in a volume of 100 µL, Cy3-conjugated 3C2 anti-GXM antibody was added to a final concentration of 8 μg/mL, and cells were incubated for 1 h with gentle rotation.Cells were pelleted at 3,000 × g for 5 min and washed twice with 1× PBS.Cells were resuspended in 100 µL, 100-µg/mL concentration of CFW was added, and cells were incubated for 20-30 min with gentle rotation.Cells were pelleted at 3,000 × g for 5 min and washed twice with 1× PBS.Cells were resuspended in a final volume of 50 µL of 1× PBS.

Capsule shedding via GXM immunoblotting
GXM immunoblotting assays were performed as previously described (25,26).Briefly, cells were inoculated into YPD and allowed to grow for 24 h.Subsequently, dilutions of 1:100 were performed, and cells were inoculated into the desired media of interest with the exception of DMEM where 1 × 10 6 cells/mL were added to 5 mL of fresh DMEM.Cells were then incubated in their respective media for 3 days at 30°C with shaking at 225 rpm.DMEM cultures were incubated at 37°C with 5% CO 2 to induce capsule formation and subsequent shedding.After 3 days, the cultures were quantified on a cell counter (Bio-Rad TC20), and the whole culture was spun down for 5 min at 3,000 × g.The top 1.5 mL was filtered through a 0.22µm filter.From this filtration step, 1 mL was heated at 70°C for 10 min to create conditioned media.Subsequent conditioned media was loaded onto a 0.6% Megabase agarose gel (Bio-Rad #1613108) and run for 8-10 h at 40 V.To normalize the amount of GXM loaded onto the gel, cell counts were taken for each strain in each medium.The amount of GXM loaded was normalized to the lowest cell count in a total volume of 100 µl.When the dye front was at the bottom of the gel, the electrophoresis was stopped, and the gel used to assemble the immunoblot sandwich.Using a Nylon membrane, the sandwich was assembled as follows, starting from the bottom: wick, three pieces of thick blotting paper, gel, membrane, three pieces of blotting paper, and paper towels (approximately 5 cm in height).The immunoblot sandwich was incubated with a 20× sodium citrate buffer for 10-12 h or until all the paper towels were saturated.The membrane was blocked for 48 h in 1× TBS-5% milk and incubated for 1 h in 1× TBST-1% milk with 1 µg/mL of anti-GXM monoclonal antibodies F12D2 and 1255.The membrane was rinsed three times in 1× TBST and incubated for 1 h in 1× TBST-1% milk with Odyssey antibody at 1:10,000.The membrane was rinsed again three times in 1× TBST and imaged on the Odyssey Imaging System (LI-COR Biosciences).

Capsule shedding via ELISA
GXM ELISA was done as described using a kit by IMMY-Labs (#CRY 101) (25).Conditioned media from cells were obtained as described above for GXM immunoblots.Conditioned media were diluted by a factor of 1 × 10 −4 or 1 × 10 −6 using serial dilutions due to the sensitivity of the assay.Lyophilized GXM was provided by the Brown Lab at the University of Utah, diluted to a concentration of 2 mg/mL, and working stocks were generated.The highest standard used in the ELISA was 78 ng/mL.

XTT biofilm assay
Assay was performed as previously described (47).Strains were grown overnight in YPD, cells were then counted (Bio-Rad TC20) and diluted to 1 × 10 7 cells/mL in DMEM, and 100 µL of the cell suspension was seeded into wells of a microtiter plate.Plates were incubated at 37°C + 5% CO 2 for 48 h, washed with 1× PBS using a microtiter plate washer (405LS, Agilent), and 100 µL of XTT/menadione solution (Sigma) was added to each well.The plates were incubated for 3 h, and 75 µL of the supernatant was removed and transferred to a new microtiter plate.Plates were read in a microtiter plate reader at 490 nm.

Uptake assay
Uptake assays were performed as previously described (8).Phorbol 12-myristate 13-acetate-differentiated THP-1 cells were incubated with Lucifer yellow-stained fungal cells that had been opsonized with 40% human serum collected from healthy donors (approved by the Notre Dame Institutional Review Board as a non-human subject research procedure).After incubation for 1 h at 37°C with 5% CO 2 , plates were washed with 1× PBS using a microplate washer (405LS, Agilent).Cells were then fixed with 4% formaldehyde and stained with DAPI (Sigma) and Cell Mask (Invitrogen).NaN 3 in PBS was added to the plates and imaged on a Zeiss Axio Observer Microscope with an automatic stage.Each well was imaged using a 3 × 3-grid set up, and resulting images were analyzed using a Cell Profiler pipeline to determine the phagocytic index (PI, # of internal cells per 100 THP-1) values.

Infections with Galleria mellonella
G. mellonella third instar larvae were sorted and weighed as described (48).Larvae weighing 200 mg and above were used in the following infection assay.Overnight cultures were grown and measured for an OD of 0.5-1.Cells were then washed twice in 1× PBS and counted using a cell counter.A total inoculum of 1 × 10 5 cells/larvae was used in a total of 5 µL.The back larval prolegs were swabbed with 70% ethanol, and 5 µL of culture was injected into the back right proleg using a Hamilton syringe (Hamilton, #80300).Syringes were sanitized and cleaned before and after each strain with both 70% ethanol and dH 2 O. Survival was assessed daily.At the time of death, infected G. mellonella larvae were placed in 3 mL of dH 2 O in a 15-mL Falcon tube.Larvae were then homogenized using a tissue homogenizer (VWR #10032-336) to break down tissue.Once sufficiently homogenized, worm slurry was then diluted 10,000× or 20,000× times and plated on YPD plates supplemented with either nourseothricin and ampicillin (AMP) or Gentecin (G418) and AMP.Plates were left to incubate for 2-3 days and then imaged on the Bio-Rad EZ Gel Doc imager for CFU quantification.

FIG 1
FIG 1 Ham1 is a palmitoylated protein conserved across the fungal kingdom.(A)A phylogenetic tree showing conserved homologs of HAM1 (CNAG_02129) across common pathogenic and model fungal species.The evolutionary history of the sequences was inferred using the maximum likelihood method with MEGAX software (13).The tree with the highest log likelihood (−11,862.20) is shown.A total of 1,000 replicate analyses (bootstraps) were run, and the percentage of trees in which the associated taxa clustered together is shown next to the branches.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.(B) Immunoprecipitation (IP) coupled with bioorthogonal click chemistry to probe palmitoylation of Ham1.Cultures of untagged (KN99) or tagged (Ham1-FLAG) cells were either not fed (-) or fed (+) an analog of palmitate (alk15) and then lysed to generate a protein extraction.An IP was then performed on fed and unfed fractions to pull down Ham1-FLAG which was then coupled to a fluorescent reporter by click chemistry, highlighting palmitoylated proteins.(Top row) A representative in-gel fluorescence experiment showing signal only in the fed fractions.(Bottom row) The accompanying western blot of the same samples run above, probed with anti-FLAG antibody.Ham1-FLAG is ~102 kDa; highlighted ladder lanes are 100 and 130 kDa.(C) Predicted palmitoylation scores of HAM1 and homologous genes used in (A), using GPS-Palm (14).Residues predicted with low confidence (scores 0.6484-0.7750)are highlighted in red.

FIG 2
FIG 2 ham1Δ mutants have higher cellular fusion efficiency and exhibit a dry colony morphology (A) The percent of colonies growing in double antibiotic plates (number of colonies that are NAT and G418 resistant over total colonies) resulting from the indicated bisexual crosses is shown.Each condition (no pheromone and with the different concentrations of exogenous pheromone) was compared to the respective WT control (KN99α x KN99a) condition by two-way ANOVA (Continued on next page)

FIG 2 (
FIG 2 (Continued)with a Dunnet's multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; N = 5-9 biological replicates depending on the cross and condition.ND, not detected.(B) Representative images of colony morphology (Colony Body) from all crosses at ×2 magnification and ×100 magnification of cells from each colony stained with CFW to highlight septal divisions of hyphal form in mutant crosses and with DAPI to show binuclear distribution in mutant crosses.Scale bars represent 500 and 10 μm, respectively.

FIG 3
FIG 3 ham1Δ unilateral and bilateral crosses exhibit increased hyphal production on V8 mating medium.(A) Representative images of WT and mutant crosses on V8 mating media.All images taken at ×63 magnification scale; bar represents 1 mm.(B) Quantification of hyphal area for all crosses at all time points.Hyphal area was calculated in FIJI (ImageJ) as the difference between a cell body mask and a mask covering the whole cell body and surrounding filaments.All mutant crosses were compared to WT controls (KN99α × KN99a) by one-way ANOVA with Dunnet's multiple comparison test; *P < 0.05, **P < 0.01.N = 3 biological replicates for all timepoints of hyphal area.

FIG 4
FIG 4 ham1Δ mutants have altered competitive fitness when compared to WT. (A) Schematic of how the cellular competition assay was performed.(B) Definition of the competitive index (CI) used to compare the fitness of strains A and B in a mixture of three strains.(C) Outcomes of the competitive assays for each of the indicated mixtures.The dashed horizontal line represents a CI of 1 or equal competition.Black circles represent the average of each independent assay, while the bar shows the mean and standard error of all assays (n = 5-7 biological replicates for all competition assays).Values were compared by one-way ANOVA with uncorrected multiple comparisons (unpaired t-tests with Welch's correction).*P < 0.05; **P < 0.01; ***P < 0.001.

FIG 5 HAM1
FIG 5 HAM1 does not regulate pheromone expression, but its expression correlates with the mating status of the cell.(A) Relative transcriptional analysis of MATα in mating conditions (V8 media) relative to non-mating conditions (YPD media) after incubation for 1, 3, 5, and 7 days on the respective media.(B) Relative transcriptional analysis of MATa in mating conditions (V8 media) relative to non-mating conditions (YPD media) after incubation for 1, 3, 5, and 7 days on the respective media.N.D., not detected.(C) Quantification of the transcript level of HAM1 in non-mating (YPD) and mating (V8) conditions after incubation for 1, 3, 5, and 7 days on each media.Each day, the YPD values were compared to the V8 values by a Mann-Whitney test.*P < 0.05.N = 5 biological replicates for all timepoints and conditions.(D) Same as (C), but measured in the indicated crosses or monoculture suspensions.For each cross or monoculture suspension, the YPD values were compared to the V8 values by a Mann-Whitney test.*P < 0.05; **P < 0.01; ***P < 0.001.N = 2 biological replicates for all timepoints and conditions.Red line shows expression levels in YPD.

FIG 6
FIG 6 ham1Δ shows no defects in major virulence mechanisms (A) Representative images of India ink staining of induced capsule in the indicated strains.Scale bar represents 10 µm.(B) Quantification of capsule size after induction.Shown is the average value; N = 25 cells for all strains.(C) Indicated strains were spotted on caffeic acid plates to visualize melanin production; lac1Δ is a melanin mutant and serves as a negative control.(D) Measurement of uptake by THP-1 phagocytic cells as a function of the number of engulfed fungi divided by the number of THP-1 cells normalized to the WT controls; pbx1Δis a known mutant with a higher uptake index, and opt1Δ is a known low uptake mutant.Results were compared using a one-way ANOVA with Dunnet's multiple comparison test for all strains with the MATα background; this includes our positive (pbx1Δ) and negative (opt1Δ) controls; *P < 0.05, ****P < 0.0001.A Student's t-test using a Gaussian distribution was used to compare phagocytic indices of KN99a and ham1Δa, as they are both in the MATa background.

FIG 7
FIG 7 ham1Δα exhibits defects in capsule attachment and transfer.(A) Representative images of WT, ham1Δα and pbx1Δ capsules stained in India ink before and after sonication.Scale bar represents 5 µm.(B) Quantification of the percentage of capsule retained post-sonication.One-way ANOVA with a Dunnet's multiple comparison test relative to WT; **P < 0.01, ****P < 0.0001, N = 20 cells for all strains.

FIG 8
FIG 8 ham1Δα exhibits increased exopolysaccharide shedding and biofilm production.(A) Representative GXM immunoblots of exopolysaccharide shedding in capsule-non-inducing media (YPD and YNB) and capsule-inducing medium DMEM.(B-D) Quantification of shed exopolysaccharide in YPD (B), YNB (C), and DMEM (D) via sandwich ELISA shown as fold change relative to WT. N.D., not detected.One-way ANOVA with Dunnet's multiple comparison test relative to WT; *P < 0.05, **P < 0.01; N = 3 for all ELISAs.(E) Biofilm formation was quantified using the XTT reduction assay, measured by absorbance at 490 nm.The cap59Δ is known to not form biofilms and used as the negative control.A one-way ANOVA with multiple comparisons was run relative to WT (KN99α); *P < 0.05; ***P < 0.001; N = 3 biological replicates.

FIG 10
FIG 10 Proposed model of HAM1 involvement in C. neoformans biology.Based on this study, we believe that HAM1 acts as a negative regulator in the mating