Widespread fungal–bacterial competition for magnesium lowers bacterial susceptibility to polymyxin antibiotics

Fungi and bacteria coexist in many polymicrobial communities, yet the molecular basis of their interactions remains poorly understood. Here, we show that the fungus Candida albicans sequesters essential magnesium ions from the bacterium Pseudomonas aeruginosa. To counteract fungal Mg2+ sequestration, P. aeruginosa expresses the Mg2+ transporter MgtA when Mg2+ levels are low. Thus, loss of MgtA specifically impairs P. aeruginosa in co-culture with C. albicans, but fitness can be restored by supplementing Mg2+. Using a panel of fungi and bacteria, we show that Mg2+ sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg2+ limitation enhances bacterial resistance to polymyxin antibiotics like colistin, which target gram-negative bacterial membranes. Indeed, experimental evolution reveals that P. aeruginosa evolves C. albicans-dependent colistin resistance via non-canonical means; antifungal treatment renders resistant bacteria colistin-sensitive. Our work suggests that fungal–bacterial competition could profoundly impact polymicrobial infection treatment with antibiotics of last resort.


Introduction
Competition is a pivotal force shaping microbial life.To thrive in crowded microbial communities, microbes have evolved a wide range of strategies to compete for resources or suppress the fitness of their neighbors, for example, by using iron scavengers [1], contact-dependent inhibition [2], or the production of antibiotics [3][4][5].Extensive studies on inter-bacterial competition have revealed profound impacts of inter-species competition on the diversity of bacterial strains, coevolution of bacterial species, and species composition within microbial communities [6].Competition between fungi and bacteria has been relatively less well studied despite the fact that fungi and bacteria cohabit many polymicrobial environments, from soils [7] to cheese [8] to host-associated niches, such as the human gut or polymicrobial infections [9].
Accumulated evidence suggests that fungi or bacteria use specific strategies to sense or combat each other.For instance, fungal-bacterial coexistence is known to drive the production of anti-bacterial [10] or anti-fungal [11] metabolites.Some bacteria produce toxins that can target certain fungal species [12,13].A recent study based on a synthetic microbial community revealed that fungi can simultaneously suppress and promote bacterial fitness [14].Fungal-bacterial competition has also been reported in some metagenomic studies [15].However, fungal-bacterial competition is hard to dissect in complex microbial communities.As a result, the underlying molecular mechanisms of fungal-bacterial competition remain largely unknown.A greater understanding of fungal-bacterial competition could reveal novel insights that enhance our ability to predict the biological outcomes of fungal-bacterial competition and help devise strategies to control fungal-bacterial interactions within diverse microbial communities.
Here, we focus on the fungus Candida albicans and the bacterium Pseudomonas aeruginosa, 2 opportunistic microbes that are frequently found together in close contact in diverse sites including in biofilms [16,17], chronic wounds [18], and the airways of people afflicted with cystic fibrosis [19,20].Previous studies have identified strategies that C. albicans and P. aeruginosa use to antagonize each other, including bacterial toxins that target fungal hyphae [17,21], interference with quorum-sensing regulation [22,23], and competition for limited resources like iron [24].Most studies have focused on anti-fungal strategies imposed by bacteria, whereas fungal strategies for competing with bacteria remain less well-understood.Similarly, it is unclear whether or how fungal-bacterial competition influences the evolution of drug resistance.Identification of strategies used by fungi to antagonize bacteria, potential bacterial counterstrategies, and the consequences of such fungal-bacterial competition on antibiotic resistance could be leveraged to predict resistance development in polymicrobial infections and improve therapies to cure infectious diseases.

Results
To identify strategies used by fungi to antagonize bacteria, we first investigated whether coculture with C. albicans (strain SC5314) affects the fitness of P. aeruginosa (strain PAO1) in brain heart infusion (BHI) broth, a medium commonly used for isolating microbes from clinical samples or studying human microbial pathogens in vitro.We found that co-culture with PAO1 did not impair C. albicans fitness, whereas co-culture with C. albicans impaired PAO1 fitness in BHI media 10-to 100-fold relative to bacteria-only conditions (monoculture) (S1A Fig) .Previous studies have investigated P. aeruginosa transcriptomes in a variety of media but not in BHI broth [25].Similarly, P. aeruginosa transcriptomes in co-culture with C. albicans have not been investigated in BHI broth [26].Therefore, to understand the physiological basis for P. aeruginosa impairment in co-culture with C. albicans in BHI media, we performed an RNA-seq analysis on P. aeruginosa following 8 h of co-culture with C. albicans, relative to monoculture.We chose this time point for the transcriptome analysis as bacterial fitness was nearly identical between the 2 conditions at this time (S2 Fig) .Our analysis revealed that 145 P. aeruginosa genes were up-regulated by at least 4-fold in co-culture relative to monoculture conditions, including those related to TonB-dependent substrate transport, siderophore synthesis, and RNA polymerase sigma factor 70 (S3A Fig and S1 Table).We also found that 134 genes, including those for Type VI secretion system, co-factor biosynthesis, and energy generation, were down-regulated by at least 4-fold in co-culture (S3B Fig and S1 Table).These changes in gene expression suggest that P. aeruginosa cells prioritized nutrient uptake while minimizing energy expenditure in co-culture with C. albicans.
We hypothesized that P. aeruginosa might rely on fungal-defense genes to protect itself during co-culture with C. albicans; loss of such fungal-defense genes would further impair P. aeruginosa fitness under co-culture conditions [27].To identify genes important for bacterial fitness in the presence of fungi, we used a transposon-insertion sequencing (Tn-seq) approach [28][29][30] to conduct a genome-wide fitness screen in bacteria.We cultured a pool of 10 5 unique P. aeruginosa transposon-insertion (Tn) mutants [28] for 10 generations, either in monoculture or in co-culture with C. albicans.We then quantified the fitness of each Tn mutant by comparing the number of reads of each transposon mutant in both conditions (Fig 1A).Our findings revealed that Tn insertions in 8 P. aeruginosa genes significantly reduced bacterial fitness in co-culture relative to monoculture conditions (Fig 1B and 1C).Three adjacent genes-PA4824, PA4825, and PA4826-showed the most significant fitness loss in co-culture (Fig 1B).These 3 genes were also the only overlap between our Tn-seq and RNA-seq analyses, with PA4824 and PA4825 showing a 32-fold increase in expression in co-culture (S3C Fig) .Of these 3 genes, only PA4825 has been functionally characterized; it encodes a magnesium transporter known as MgtA [31].In addition to the 8 genes in which Tn insertions decreased fitness (putative fungal-defense genes), our Tn-seq analyses identified 18 genes whose loss enhanced bacterial fitness in co-culture (S2 Table ), suggesting these genes either become "dispensable" or incur a fitness cost in co-culture conditions.

Magnesium is an axis of nutritional competition between P. aeruginosa and C. albicans
We focused on the contributions of the PA4824, PA4825 (mgtA), and PA4826 genes during coculture.We engineered gene deletion mutants and measured the fitness of these mutants relative to a fluorescently labeled wild-type (WT) strain in either monoculture or co-culture with C. albicans using competitive fitness assays, which resemble the pooled Tn-seq bulk selection conditions (S4A Fig) .Our experiments showed that the relative fitness of the ΔPA4824 or ΔPA4825 (ΔmgtA) mutants, but not ΔPA4826, was approximately 10% lower than the WT in co-culture (S4B Fig) .In contrast, neither of these mutants had any measurable fitness defect, relative to WT PAO1, in monoculture (S4B Fig) .Co-culture colony formation assays also confirmed our findings; deletion of either ΔPA4824 or ΔmgtA led to a 10-fold reduction in colonyforming units (CFUs) of P. aeruginosa in co-culture relative to the CFUs of WT in co-culture (Fig 1D).CFUs of ΔPA4824 or ΔmgtA in co-culture were restored by expressing a WT copy of PA4824 or mgtA in trans (S4C and S4D Fig) .Moreover, a double deletion ΔPA4824 ΔmgtA mutant was even more significantly impaired than single deletion mutants (Fig 1D).These results suggest that the protein products encoded by PA4824 and mgtA are independently required for optimal P. aeruginosa fitness under co-culture conditions, but PA4826 is not.We infer that the identification of PA4826 in our Tn-seq analyses may be due to the effects of insertional mutants in PA4826 on the expression of adjacent PA4824 and PA4825 (mgtA) genes.
Our finding that P. aeruginosa PA4825 (mgtA) is required for full bacterial fitness in co-culture led us to hypothesize that C. albicans depletes the vital Mg 2+ cation in co-culture.Under Mg 2+ depletion conditions, mgtA might become essential for fitness since its loss would result in lower intracellular Mg 2+ levels.To test this hypothesis, we employed a Mg 2+ genetic sensor [31] from Salmonella enterica serovar Typhimurium.Previous studies showed that S. Typhimurium mgtA expression levels are controlled by its 5 0 untranslated region (5 0 UTR), which acts as a ribo-switch, adopting different stem-loop structures depending on the Mg 2+ levels and regulating the transcription of mgtA [31].We showed that this S. Typhimurium Mg 2+ sensor can accurately detect changes in intracellular P. aeruginosa Mg 2+ levels (S5 Fig) .Using this sensor, we found that the intracellular Mg 2+ levels in the WT P. aeruginosa are reduced by 2-fold in C. albicans-spent media (i.e., derived from the filtrate of C. albicans cultures to simulate Mg 2+ depletion) compared to in fresh BHI media (Fig 1E).Loss of mgtA further reduced bacterial intracellular Mg 2+ levels in the fungal spent media, but not in monoculture conditions (Fig 1E).Surprisingly, loss of PA4824 also reduced intracellular Mg 2+ levels in fungal spent media compared to the WT strain, with a double deletion ΔPA4824 ΔmgtA mutant further reducing intracellular Mg 2+ levels, compared to the single gene deletion mutants (Fig 1E).These experiments confirm fungal-imposed Mg 2+ sequestration and demonstrate that mgtA and PA4824 are independently required for both maintaining intracellular P. aeruginosa Mg 2+ levels as well as fitness of P. aeruginosa cells in co-culture with C. albicans.
Our findings suggest that PA4824 may play a crucial role in Mg 2+ uptake.Although its function remains uncharacterized, Alphafold structural predictions [32] revealed that PA4824 likely encodes a transmembrane protein with a distinctive β-barrel structure commonly associated with proteins involved in ion transport or nutrient uptake (S6A Fig) .The core of PA4824 is composed of hydrophilic and negative-charged residues (S6B and S6C Fig) , suggesting it might transport positive-charged molecules.Based on our co-culture RNA-seq and Tn-seq  experiments, results from the Mg 2+ genetic sensor assay, and the Alphafold prediction of PA4824 protein structure, we speculate that PA4824 potentially acts as a novel Mg 2+ transporter.
In addition to MgtA (and potentially PA4824), P. aeruginosa uses 2 other constitutively expressed Mg 2+ transporters, CorA and MgtE, to obtain Mg 2+ from the environments [33].However, neither CorA nor MgtE was implicated in competition with C. albicans by our Tnseq or RNA-seq analyses (S7A Fig) .We also found that corA or mgtE loss-of-function mutants did not alter P. aeruginosa fitness in co-culture conditions (S7B Fig), nor did they significantly reduce intracellular Mg 2+ levels in C. albicans-spent BHI compared to ΔmgtA mutant (S7C Fig) .Our experiments reveal that MgtA is the primary Mg 2+ transporter induced in co-culture, crucial for bacterial fitness and Mg 2+ uptake in co-culture.Thus, we conclude that that MgtA is a key bacterial Mg 2+ transporter required to overcome fungal sequestration of Mg 2+ .
If C. albicans sequesters Mg 2+ from P. aeruginosa, we hypothesized that supplementing cultures with Mg 2+ might rescue the fitness defects of the ΔmgtA and ΔPA4824 mutants.Indeed, supplemental Mg 2+ (10 mM) was sufficient to restore the fitness of ΔmgtA, ΔPA4824, and ΔPA4824 ΔmgtA double deletion mutants to WT levels in co-culture conditions (Fig 1F).To investigate whether Mg 2+ could explain fitness effects of other candidate genes revealed by the Tn-seq analyses, we repeated our Tn-seq experiments in monoculture versus co-culture conditions except that we added 10 mM Mg 2+ to BHI media.To our surprise, we found that Mg 2+ supplementation ameliorated the fitness effects of most of the candidate genes identified in our initial Tn-seq analyses (S8A Fig), including all 8 fungal-defense genes and 17 of 19 dispensable genes crucial to fundamental cellular processes, including cell division, pyrimidine synthesis, and energy production-enzymes in essential processes that rely on Mg 2+ .Despite their importance to bacterial physiology, we hypothesize that these otherwise critical processes might become too costly to maintain in low Mg 2+ co-culture conditions.Only 2 genes (PA4029 and PA5484) remained marginally essential in co-culture, even under supplemented Mg 2+ conditions (S8A Fig) .Notably, Mg 2+ supplementation only affected the expression of PA4824-PA4826, but not other candidate genes (S8B Fig) , suggesting that Mg 2+ levels affect their functions posttranscriptionally.Thus, our finding underscores the influential role of Mg 2+ competition in mediating the fitness effects of most candidate genes in co-culture conditions.We note, however, that Mg 2+ supplementation did not fully restore the fitness of P. aeruginosa WT cells in co-culture to monoculture fitness levels (Fig 1F ), consistent with prior observations of additional, Mg 2+ -independent, axes of antagonism between bacteria and fungi [26,34,35].
Nutritional competition for important metal ions, such as iron, zinc, and manganese, is known for competition among microbes and between microbes and vertebrate hosts [36][37][38].However, competition for the vital Mg 2+ cation is a novel finding.We reasoned that our finding might reflect different Mg 2+ levels in various media conditions.Indeed, we found that MgtA is required for P. aeruginosa fitness in co-culture with C. albicans in BHI and tryptic soy broth (TSB) media (S9A Fig) , but not in yeast extract-peptone-dextrose (YPD) or synthetic cystic fibrosis media (SCFM) [39] .Additionally, by titrating Mg 2+ levels in co-culture, we found that supplementation of BHI with greater than 300 μm Mg 2+ was sufficient to relieve the fitness defect of the P. aeruginosa ΔmgtA mutant (S9F Fig) .Thus, we conclude that BHI media represents a low Mg 2+ condition that reveals a novel mode of nutritional competition between fungi and bacteria.
MgtA was previously described as a Mg 2+ -specific transporter [31].To rule out the possibility that the fitness impairment of ΔmgtA mutant in co-culture could be caused by the reduction of other cations in BHI, we performed a series of supplementation experiments with alternate cations.We found that Mg 2+ levels specifically determine P. aeruginosa viability in co-culture with C. albicans; supplementation of zinc (Zn 2+ ) or copper (Cu 2+ ) ions did not restore the viability of ΔmgtA mutant in co-culture to the monoculture levels (S9G Fig) .The addition of molybdenum (Mo 6+ ) or manganese (Mn 2+ ) ions drastically decreased P. aeruginosa viability even in monoculture (S9G Fig), which rules out the possibility that either of these 2 ions was limited in BHI.Finally, we found that only a very high concentration of Ca 2+ (10 mM) rescued the fitness defect of the ΔmgtA mutant in co-culture (S9H Fig), orders of magnitude higher than the concentration of Mg 2+ required to rescue ΔmgtA fitness in co-culture (S9F Fig) .These experiments further confirm that the fitness of ΔmgtA in co-culture conditions is primarily determined by Mg 2+ sequestration.

Widespread Mg 2+ competition between fungi and gram-negative bacteria
We next assessed whether Mg 2+ competition is a general means of antagonism by fungi against bacteria.Escherichia coli and S. Typhimurium are gram-negative bacteria that coexist with C. albicans in the human gut [40].E. coli encodes a single mgtA ortholog (like P. aeruginosa), whereas S. Typhimurium encodes 2 paralogs: mgtA and mgtB.We found that either a single deletion ΔmgtA in E. coli or a double deletion ΔmgtA ΔmgtB in S. Typhimurium severely reduced bacterial fitness in co-culture with C. albicans, and such fitness reduction was restored via Mg 2+ supplementation (Fig 2A and 2B).
Next, we expanded our analyses to 3 additional fungal species that coexist with P. aeruginosa in polymicrobial infections [41]: Candida tropicalis, Candida parapsilosis, Candida glabrata, as well as a non-pathogenic species, Saccharomyces cerevisiae.In each case, we found that the P. aeruginosa ΔmgtA mutant had reduced fitness relative to WT cells in fungal co-culture (Fig 2C), which could be restored by Mg 2+ supplementation (Fig 2D).
This mode of competition might be highly specific between fungi and diverse gram-negative γ-proteobacteria we have tested; it does not manifest in inter-bacterial competition.For example, lack of mgtA did not lower P. aeruginosa fitness in co-culture with bacteria that commonly co-exist in CF airways, such as gram-positive Staphylococcus aureus (Fig 2E) or gramnegative Burkholderia multivorans (Fig 2F).Similarly, P. aeruginosa ΔmgtA mutant had similar fitness as WT cells in co-culture with other gut-colonizing gram-negative like E. coli or S. Typhimurium (Fig 2F).Despite BHI representing a low-Mg 2+ condition, our results showed that different bacterial species do not necessarily compete for Mg 2+ during co-culture, suggesting fungi may have a higher demand or propensity for Mg 2+ uptake and sequester Mg 2+ more effectively than gram-negative bacteria.Overall, our findings confirm that competition for Mg 2+ is a widespread mode of fungal antagonism against gram-negative bacteria and that MgtA plays an important role in counteracting fungal-mediated Mg 2+ competition.Whether fungi can suppress gram-positive bacteria through the same mechanism of Mg 2+ competition remains an open question.
How do fungi impose competition for Mg 2+ ?We investigated whether the loss of S. cerevisiae genes in Mg 2+ uptake or sequestration restores the fitness of P. aeruginosa ΔmgtA mutant.S. cerevisiae cells store 80% of cellular Mg 2+ in their vacuole [42].To investigate whether Mg 2+ sequestration in the fungal vacuole is involved, we generated a deletion mutant in Mnr2 [43], previously proposed to be a vacuolar Mg 2+ transporter in S. cerevisiae.We found that the fitness of P. aeruginosa ΔmgtA mutant was restored to WT levels in co-culture with the mnr2Δ mutant (Fig 2G).Yet, S. cerevisiae mnr2Δ mutant had similar fitness as WT S. cerevisiae both in media only as well as in co-culture (S10 Fig), suggesting that lack of Mnr2 does not cause a strong growth defect.In contrast, P. aeruginosa ΔmgtA fitness was not restored by competition with S. cerevisiae mutants lacking Alr2, a cell-wall-associated Mg 2+ transporter used to acquire environmental Mg 2+ into the cytosol [44], or those lacking Mrs2/Mme1, mitochondria-specific Mg 2+ transporters (Fig 2G) [45].Thus, fungal vacuolar Mg 2+ sequestration might broadly mediate Mg 2+ antagonism against gram-negative bacteria.
To test this possibility, we grew P. aeruginosa in fresh BHI media or C. albicans-spent BHI media, which mimics Mg 2+ depletion by fungi, and examined bacterial survival when treated with colistin (also referred to as polymyxin E) (Fig 3B).Consistent with our hypothesis, we found that cells grown in fresh media showed no survival after colistin treatment, whereas 20% of cells grown in fungal spent media survived colistin treatment (Figs 3C and S11A).Supplementation with extra Mg 2+ in C. albicans-spent media restored the sensitivity of P. aeruginosa cells to colistin, to levels resembling sensitivity in BHI fresh media (Figs 3C and S11A).Since low Mg 2+ conditions activate the PmrAB and PhoPQ signaling pathways required to modify LPS and confer colistin resistance, we tested whether pmrB or phoQ loss-of-function transposon mutants [54] would abolish the observed colistin resistance in fungal spent media; we found that this was the case (Fig 3C ).
We found that fungi-dependent increased colistin survival is not restricted to P. aeruginosa or colistin.For example, we found that Mg 2+ depletion by C. albicans also increased the survival of S. Typhimurium to colistin, a phenotype that is also suppressed by extra Mg 2+ (Figs 3D  and S11B).Furthermore, Mg 2+ depletion increased P. aeruginosa survival to another polymyxin antibiotic, polymyxin B (S12 Fig) .Thus, fungal sequestration of Mg 2+ provides a general means of conferring polymyxin resistance on gram-negative bacteria.
Since fungal spent media cannot fully recapitulate fungal presence in co-culture conditions, we tested whether fungal co-culture also conferred increased colistin survival (Fig 3E).We found that co-culture with C. albicans was sufficient to protect P. aeruginosa and S. Typhimurium from colistin across a range of concentrations (1.5 μg/ml to 12.5 μg/ml; the clinical MIC of P. aeruginosa to colistin is 2 μg/ml), in contrast to monoculture conditions at both 30 ˚C (Figs 3F, 3G, S13A and S13B) and 37 ˚C (S14A and S14B Fig) .Additionally, the protection from antibiotics by fungi in co-culture applies to different P. aeruginosa strains.We found that co-culture with C. albicans also protects several colistin-sensitive P. aeruginosa isolates from cystic fibrosis patients from colistin (S15 Fig) . Thus, C. albicans can protect gram-negative bacteria from colistin across a range of conditions.significantly different from the WT strain (orange) in monoculture.However, the relative fitness of P. aeruginosa ΔmgtA was impaired in co-culture with either WT S. cerevisiae or either of 3 S. cerevisiae mutant strains: Δalr2, Δmrs2, or Δmme1.In contrast, the relative fitness of P. aeruginosa ΔmgtA was restored in co-culture with S. cerevisiae Δmnr2 strain.Mean ± std of 4    2+ , and then passaged until exponential phase, when they were treated with 25 μg/ml colistin.Bacterial survival is shown as the percentage of viable cells after the colistin treatment.(C) WT P. aeruginosa strains were highly susceptible to colistin treatment in BHI media.However, they could survive colistin treatment in C. albicans-spent BHI media but not in spent BHI media supplemented with 10 mM Mg 2+ .In contrast to WT strains, pmrB or phoP Tn-mutants of P. aeruginosa (which cannot modify LPS) remained highly susceptible to colistin

Fungal co-culture fundamentally alters the evolution of bacterial colistin resistance
Understanding how microbial interactions drive the evolution of antibiotic resistance is important for biological and biomedical reasons; increased antibiotic resistance is often observed in polymicrobial communities [55].We hypothesized that long-term coexistence with C. albicans may fundamentally alter the onset and evolution of enhanced colistin survival in P. aeruginosa.To test this possibility, we passaged 8 replicate WT populations of P. aeruginosa PAO1 for 90 days, either in monoculture or in co-culture with C. albicans, with each population exposed to gradually increasing concentrations of colistin (from 1.5 μg/ml to 192 μg/ ml) (Fig 4A).As controls, we also passaged mono-and co-culture populations in the absence of colistin (Fig S16).After evolution, both monoculture-and co-culture-evolved populations gained increased survival to 192 μg/ml colistin (Fig 4B).Surprisingly, we found that enhanced colistin survival in P. aeruginosa co-culture populations was dependent on C. albicans; colistin survival was almost completely lost upon removal of C. albicans using antifungal treatment or the addition of Mg 2+ (Figs 4C and S17).Our findings indicate that co-culture-evolved populations acquire increased colistin survival due to fungal protection induced by Mg 2+ sequestration but continue to depend on the fungus for increased protection.
Based on our findings, we suspected that P. aeruginosa populations evolved in co-culture with fungi likely enhance colistin survival via fundamentally different mutational mechanisms than monoculture-evolved populations.To test this hypothesis, we performed genome sequencing of all experimentally evolved populations.We found that all colistin-treated populations grown in monoculture became hypermutators (Fig 4D) by acquiring at least 1 mutation in genes involved in DNA replication or repair (uvrB, recQ, and mutS) (Fig 4E and S3 Table).Hypermutation is a hallmark of antibiotic-resistance acquisition that has been observed in several previous experimental evolution studies [56].As a result of hypermutation, each monoculture population acquired hundreds of fixed mutations (Fig 4D ), including mutations previously shown to confer colistin resistance, in genes such as pmrB [57], ptsP [58,59], pqsR [58], colS [60,61], porins [58], and proteins related to bacterial motility [62] (Fig 4E and S3 Table ).In contrast, none of the co-culture-evolved populations became hypermutators; each only harbored 6 to 8 fixed mutations (Fig 4D ), primarily in other targets for colistin resistance, such as genes in LPS biosynthesis [63] and an outer-membrane protein oprH [52] (Figs 4E and S18 and S3 Table ).Intriguingly, we found that PA4824, another co-culture-specific defense gene identified in our Tn-seq experiments, was also mutated in all 8 replicates of co-cultureevolved populations (Figs 4E and S14 and S3 Table).We did not find fixed mutations in any of these genes in co-culture populations that were not exposed to colistin (S4 Table ).Thus, the dual stressors of colistin and fungal co-culture specifically select for distinct classes of adaptive mutations, fundamentally altering the mode of bacterial colistin survival.even in C. albicans-spent BHI media.(D) WT S. Typhimurium was highly susceptible to 3 μg/ml colistin in either BHI media or C. albicans-spent media supplemented with 10 mM Mg 2+ but became more resistant to colistin in fungal spent media alone.(E) Schematic of the colistin survival assay in co-culture.The P. aeruginosa WT strain was cultured only in BHI or co-culture with C. albicans for 18 h.Monocultures or co-cultures were then treated with colistin.Bacterial survival is calculated as the percentage of viable cells after the colistin treatment, just as in (B).(F) P. aeruginosa co-cultures with C. albicans (orange) were more resistant than monocultures (blue) over a series of colistin concentrations, from 1.5 μg/ml colistin (clinical MIC) to 12.5 μg/ml colistin (8× clinical MIC).

Discussion
By investigating bacterial defense strategies against fungi, we discovered that Mg 2+ is a novel, general axis of nutritional competition between fungi and gram-negative bacteria (Fig 5).Under low Mg 2+ conditions, diverse gram-negative bacteria such as P. aeruginosa, E. coli, and S. Typhimurium rely on a specific Mg 2+ transporter, MgtA, to overcome fungal-mediated Mg 2+ sequestration.Despite reducing bacterial fitness, such Mg 2+ sequestration also shields bacteria from polymyxin antibiotics used for treating multi-drug resistant gram-negative bacteria while impeding the genetic evolution of colistin resistance (Fig 5).Overall, our study uncovers pivotal but previously unexplored connections between nutritional competition, antibiotic survival, and microbial evolution.
Our study indicates that fungal-bacterial competition for Mg 2+ likely occurs in conditions with Mg 2+ levels lower than approximately 0.45 mM, a threshold that aligns with lower Mg 2+ conditions observed during P. aeruginosa infection in humans [64].In addition to fungal sequestration, physiological availability of Mg 2+ can be affected by various cation-chelating agents during infection, including citrates produced by other bacteria [65], or extra-cellular DNA in biofilms [66], or macrophage-induced Mg 2+ sequestration [67].Low serum Mg 2+ levels are also associated with other polymicrobial infectious diseases, like diabetic foot ulcers [68] and urinary tract infections [69].However, understanding microbial access to Mg 2+ in polymicrobial infections under physiological conditions remains challenging.Although half of cystic fibrosis patients suffer from hypomagnesemia (serum Mg 2+ levels < 0.75 mM) [70,71], the physiological Mg 2+ levels in their lungs or sputum are understudied.The few reports of Mg 2+ levels in CF sputum from clinical studies range from 0.49 ± 0.32 mM [64] to 0.78 to 1 mM [72,73].This range could reflect differences in patient conditions, sampling methods, or Mg 2+ quantification techniques.The complexity of CF environments further complicates assessing Mg 2+ availability for microbes during infections.Moreover, measurements of Mg 2+ levels based on sputum samples may not reflect the bioavailability of magnesium in different host microenvironments.Despite this uncertainty, molecular evidence suggests that P. aeruginosa may indeed experience Mg 2+ depletion in CF airways.Several P. aeruginosa genes that are responsive to low Mg 2+ [74], including mgtA [75], are induced in CF sputum.Other gramnegative bacteria, including S. Typhimurium, also encounter reduced Mg 2+ concentrations upon infection of hosts [67].We propose that using microbial Mg 2+ genetic biosensors in animal infection models would be a logical next step to determine bioavailable Mg 2+ levels to gram-negative bacteria during infection.
Although low Mg 2+ was previously shown to affect colistin resistance in gram-negative bacteria [52,53], our study provides the first molecular evidence that fungal-bacterial competition for Mg 2+ may profoundly impact both bacterial fitness and colistin survival.Our study reveals context-dependent fitness effects of Mg 2+ competition, which incurs general fitness costs to bacteria but provides fitness benefits in terms of colistin resistance.This tradeoff might favor polymicrobial infections, either in treatment conditions involving polymyxins or when host immune cells produce Mg 2+ -dependent antimicrobial peptides that act upon bacteria.Future exploration into how Mg 2+ competition alters other antibiotic or antifungal resistance will broaden our understanding of how polymicrobial infections influence the onset of drug resistance.Mg 2+ has been implicated in altering the susceptibility of gram-negative bacteria to antibiotics other than colistin.For instance, in S. Typhimurium, impaired mgtA or Mg 2+ homeostasis increases susceptibility to cyclohexane or nitrooxidative stress [76,77].Our study highlights the importance of studying how Mg 2+ homeostasis broadly impacts antimicrobial resistance in gram-negative bacteria.
Our evolution experiments provide novel insights into how co-habitation with C. albicans can influence the evolution of P. aeruginosa.The fungal-and Mg 2+ -dependent colistin survival we observed has crucial biomedical implications.Disrupting this fungal protection, either via concomitant antifungal therapy or via Mg 2+ supplementation may enhance the efficacy of colistin treatment or even rescue it even after colistin resistance has been apparently acquired.Consistent with this idea, hypomagnesemia in various chronic infections exacerbates disease progression, whereas oral Mg 2+ supplementation alleviates it [78,79].
The rise of colistin-resistant bacteria in environments is an escalating concern [80,81].Our discovery of genetic determinants for fungal-bacterial competition paves the way to investigating whether fungi and bacteria compete for Mg 2+ during polymicrobial infections, what the consequences of such competition are to microbes and hosts, and how fungal-bacterial interactions alter microbial drug resistance in vivo.Further investigations using P. aeruginosa clinical isolates with a history of C. albicans co-infection will extend our in vitro findings to clinical contexts and illuminate the role of Mg 2+ or other factors in shaping fungal-bacterial interactions and drug resistance during co-infection.In addition to polymicrobial infections in animals, low Mg 2+ is associated with exacerbation of plant infectious diseases [82].Since our study finds that Mg 2+ competition is widespread between fungi and gram-negative bacteria, it also hints at the exciting possibility of manipulating environmental Mg 2+ levels or fungal-bacteria interactions to constrain infectious diseases in diverse eukaryotic hosts, as well as mitigating the spread of colistin resistance.Overall, our findings reveal a novel dimension in nutritional competition and highlight the transformative potential of fundamental discoveries in guiding effective strategies to treat intractable polymicrobial infections or mitigate antibiotic resistance.

Bacterial and fungal strains and culture conditions
All bacterial and fungal strains used in this study are listed in S5 Table in the supplemental material.Bacterial strains in this study were derived from P. aeruginosa PAO1, E. coli BW25113, S. Typhimurium 14028S, S. aureus SA113, and B. multivorans AMT17616.Yeast strains were derived from C. albicans SC5314, S. cerevisiae W303.C. tropicalis CBS94, C. parapsilosis CBS604, and C. glabrata CBS562 were purchased from ATCC.Individual P. aeruginosa Tn mutant strains were obtained from Dr. Colin Manoil's lab [28,54]; correct transposon insertions were confirmed by PCR and Sanger sequencing.Clinical P. aeruginosa isolates from CF patients were obtained from Dr. Pradeep Singh's lab.Unless otherwise specified, all experiments were performed in Brain Heart Infusion Broth Media (BHI, Sigma-Aldrich), buffered with 10% MOPS to pH 7.0 followed by filter sterilization.All strains were grown in BHI and incubated at 30 ˚C or 37 ˚C.Other media used in this study were YPD (1% yeast extract, 2% peptone, and 2% D-glucose), SCFM [39], and TSB (Sigma-Aldrich).For antibiotics used in this study, 100 μg/ml gentamicin was used to select against gram-negative bacteria, and 50 μg/ ml nystatin was used to select against Candida species and S. cerevisiae.Colistin and polymyxin B (Sigma-Aldrich) were prepared as 10 mg/ml and 5 mg/ml stock solutions, respectively.

Generation of P. aeruginosa mutants
For generating gene deletions in P. aeruginosa, a pEXG2-mediated allelic exchange method was used [83].In short, pEXG2 deletion constructs were transformed into E. coli S17.S17 donor was subsequently mixed with P. aeruginosa recipients on an LB agar plate at a 5:1 ratio of donor to recipient cells, and the cell mixture was incubated at 30 ˚C overnight.The cell mixture was then scraped, resuspended into 200 μl LB, and plated on LB agar plates with 100 μg/ ml gentamicin to select for cells containing the deletion plasmid integrated into the P. aeruginosa genome.P. aeruginosa merodiploid colonies were streaked on LB-no salt-agar plates with sucrose.Gentamicin-sensitive and sucrose-resistant colonies were screened for allelic replacement by colony PCR, and gene deletions were confirmed by Sanger sequencing of PCR products.Primers and plasmids used for strain constructions are listed in S6 Table.

Generation of S. cerevisiae mutants
S. cerevisiae gene deletion mutants were generated by homologous recombination at endogenous gene loci.Gene deletion fragments were amplified from pFA6a-natMX4 and fused with 500 bp upstream and downstream sequences of the targeted coding sequence by PCR.The PCR fragment with homology arms was transformed into S. cerevisiae cells with the standard LiOAc-based protocol [84].Gene deletions were confirmed by colony PCR and Sanger sequencing to check the absence of endogenous genes.Primers and plasmids used for strain constructions are listed in S6 Table.

Fungal-bacterial co-culture CFU assay
All experiments began with bacterial or fungal cultures that were grown overnight.The starter cultures were diluted either 1:100 (for bacterial cultures) or 1:50 (for fungal cultures) in BHI and cultured for approximately 4 to 5 h to reach the log phase.Refreshed bacterial and fungal strains were added to BHI to reach a final bacterial cell density of 2.5 × 10 4 cells/ml and a fungal cell density of 5 × 10 5 cells/ml, for the co-culture experiment.The same amounts of bacterial or fungal cells were also added separately in fresh BHI, as monoculture controls.These cultures were incubated for 18 h at 30 ˚C or 37 ˚C with shaking.We chose 30 ˚C for the initial co-culture assays for 2 reasons.First, C. albicans cells reached higher CFU at 30 ˚C than 37 ˚C, which would impose a stronger competition with bacteria.Second, C. albicans cells form hyphae at 37 ˚C, which can have multiple cells in one filament and thus confound CFU measurements.We further confirmed that our findings of Mg 2+ competition are independent of temperature by setting up co-culture assays at both 30 ˚C and 37 ˚C.Cultures were serially diluted and spotted on LB plates with 50 μg/ml nystatin for measuring bacterial growth and on YPD plates with 100 μg/ml gentamicin for measuring fungal growth.

PA4824 or mgtA complementation assay
To complement the deletion of mgtA, ΔmgtA was transformed with a genome-integrated copy of mgtA under its endogenous promoter (miniTn7-mgtA) [85] or a plasmid copy of mgtA under an arabinose-inducible promoter (pBAD) [86].As PA4824 is in the same operon as mgtA, we used pBAD to express PA4824 to ensure its full expression.These deletion-complemented strains were prepared in the same way as the co-culture CFU assay, except that 0.2% arabinose was used to induce PA4824 or mgtA expression.

Bacterial-bacterial co-culture CFU assay
Overnight bacterial cultures were diluted 1:100 in BHI and cultured for approximately 4 to 5 h to reach log phase.Log-phase cultures of bacterial competitors (S. aureus, S. multivorans, E. coli, or S. Typhimurium) were added in fresh BHI at 5 × 10 5 cells/ml as final cell density while 2.5 × 10 4 cells/ml of log-phase P. aeruginosa culture was added.The same number of bacterial cells were also added separately in fresh BHI, as monoculture controls.These cultures were incubated for 18 h at 30 ˚C with shaking.Cultures were serially diluted and spotted on Pseudomonas Isolation Agar (Millipore-17208) to measure P. aeruginosa CFUs.

Measurements of bacterial intracellular Mg 2+
Cytosolic Mg 2+ concentrations in P. aeruginosa were measured using a modification of the previously reported Mg 2+ genetic sensor assay [31].First, we cloned the Mg 2+ genetic sensor containing S. Typhimurium mgtA leader sequence (P lac -mgtA leader -lacZ) or the promoter-only fragment (P lac -lacZ) into a P. aeruginosa replicating plasmid, pBBR [87].Then, these 2 plasmids were transformed into the indicated P. aeruginosa strains separately, with the former acting as a reporter for Mg 2+ and the latter acting as a control.Both strains were cultured in BHI or fungal spent media (80% v/v final concentration) containing 100 μg/ml gentamicin for 16 h and then diluted 1:100 in the corresponding media with gentamicin for 4 h to reach log phase.The cell density of these cultures was adjusted to OD600 = 0.4, and we followed the same protocol to measure β-galactosidase activity using Galacto-Light Plus System (Thermo Fisher).Finally, cytosolic Mg 2+ concentrations were derived from the following equation, where the control strain was used to normalize transcription or translation efficiency between strains: ½Mg 2þ � ¼ miller units from the control strain ð Þ= miller units from the reporter strain ð Þ

Colistin survival assay in C. albicans-spent media
To prepare C. albicans-free supernatant, a single C. albicans colony was inoculated in 10 ml BHI and cultured at 30 ˚C for 24 h.The fungal culture was centrifuged, and the supernatant was filtered with a Steriflip unit with a 0.22 μm Millipore Express PLUS membrane (Millipore-Sigma), and 8 ml fungal filtrate was added to 2 ml fresh BHI (80% v/v final concentration) to prepare C. albicans-spent media, mimicking nutrient exhaustion by C. albicans.To perform colistin treatment, we first diluted overnight bacterial culture in 3 ml BHI or C. albicans-spent media at 1:100 for 4 h.Then, bacterial cultures were adjusted to OD600 = 0.3 in BHI or C. albicans-spent media.Each bacterial culture was treated with colistin at the indicated concentration, and the samples were incubated at 30 ˚C for 1.5 h.To assay bacterial viability, bacterial cultures after colistin treatment were serially diluted on LB plates.Bacterial cultures in the same growth condition without colistin treatment were used as a control.Bacterial survival was calculated by the ratio of bacterial CFUs, relative to those observed without treatment, at 1.5 h.

Colistin survival assay in co-culture
Log-phase bacterial cells were cultured in BHI alone or in co-culture with fungal cells in BHI for 18 h at 30 ˚C or 37 ˚C, as the co-culture CFU assay indicated.To equilibrate bacterial cell numbers in these 2 culturing conditions prior to the colistin treatment, the cell density of monoculture samples was adjusted to OD600 = 0.3 to match the bacterial cell density after 18-h growth in co-culture.Both equilibrated monoculture and co-culture samples were split into 1 ml in 2 different tubes for colistin-treated versus untreated conditions.Colistin was added to the first tube at the indicated concentration.Bacterial cells in both tubes were incubated at 30 ˚C for 1.5 h and the bacterial viability was determined by counting CFUs after serial dilution.Bacterial survival was determined by the ratio of bacterial CFUs upon colistin treatment, relative to without treatment (average of 4 replicates).Bacterial survival was set to 100% if it was over 100% due to stochastic variation in colony counts.We used the same fungal protection assay to measure colistin resistance in co-cultureevolved populations.These evolved populations were split into 2 culturing conditions: BHI only, as co-culture conditions, or BHI + 50 μg/ml nystatin, to remove fungal cells (as monoculture conditions).Cells were grown for 18 h at 37 ˚C.After overnight growth, populations in both conditions were treated with 192 μg/ml colistin following the above protocol.To measure bacterial viability after colistin treatments, cells in co-culture and monoculture conditions were spotted on LB + 50 μg/ml nystatin or LB plates, respectively.

Competitive fitness assay
A WT PAO1 strain with chromosomally integrated miniTn7-mCherry was used as the reference strain in fitness competition with deletion mutants, either in BHI only or in co-culture with C. albicans.All bacterial and yeast strains were incubated in BHI at 30 ˚C to log phase, and cell density was quantified by OD600 using a spectrophotometer (biowave CO8000 Cell Density Meter).First, a non-fluorescent strain, either the PAO1 wild-type or deletion mutants, was mixed with the fluorescence-labeled reference strain at a ratio of 1:1 to make a bacterial mixed culture with 2 × 10 6 cells/ml.Part of the cell mixture was used to determine the initial ratio of sample and reference strains using flow cytometry (BD FACSymphony A5 Cell Analyzer).Then, 10 μl of cell mixture was inoculated separately in 3 ml BHI alone (monoculture) or 3 ml BHI with 2 × 10 5 C. albicans cells (co-culture) and grown in BHI at 30 ˚C for 18 h.After 18 h, monoculture samples were diluted 100-fold to measure the ratio of sample and reference strain.For co-culture samples, a low-speed spin (1,000xg for 3 min) was applied to separate bacterial and fungal populations.Supernatants enriched with bacterial cells were measured using flow cytometry.For each sample, at least 30,000 cells were collected and the FACS data were analyzed by the FlowJo 10.4.1 software.The reference strain was cultured separately to estimate the number of generations during the experiment.Each experiment was conducted in at least 2 biological and 2 technical replicates.To calculate the relative fitness, w, of each sample strain to the reference strain, we followed the formula: w = 1+s, where selection coefficient s = [ln(sample/reference) t -in(sample/reference) t = 0 ]/t, where t=number of generations and (sample/reference) is the ratio between a sample strain and the reference strain [88].

RNA sequencing and analysis
A log-phase P. aeruginosa WT culture (OD600 = 0.1~0.2) was diluted at 5 × 10 6 cells in 100 ml media alone or 100 ml media with 5 × 10 7 C. albicans cells and grown at 30 ˚C.Cells were collected for extracting RNA at 8 h when there was no significant growth difference of P. aeruginosa between monoculture and co-culture.Prior to RNA extraction, a slow-speed spin was applied to co-culture samples to enrich bacteria in the supernatant.For each sample, total RNA was harvested from 10 9 cells, preserved in RNA-protected QIAzol, and purified by RNAeasy mini kit (Qiagen) following the commercial protocol.RNA was quantified by Qubit fluorometric quantitation system (Thermo Fisher), and RNA integrity was examined by 4200 TapeStation System (Agilent).We generated an rRNA depleted library and obtained >20 million 150 bp pair-end Illumina NovaSeq reads for each sample commercially (Azenta).Reads were examined using FastQC (Barbaham Bioinformatics, Babraham Institute) and trimmed by Trim Galore! (v0.6.10;https://github.com/FelixKrueger/TrimGalore).Reads were mapped to the P. aeruginosa PAO1 genome (GCF_00006765.1) using Subread/FeatureCounts (https:// github.com/ShiLab-Bioinformatics/subread).Differential gene expression was analyzed with DESeq2 [89] (https://bioconductor.org/packages/release/bioc/htmL/DESeq2.htmL),following the default pipeline of data normalization.A log 2 fold change cutoff of 2 and a false discovery rate (FDR) of 0.05 were used to filter significantly differentially expressed genes between samples from 3 biological replicates.Gene Ontology analysis was done using the P. aeruginosa STRING database on ShinyGO 0.77(http://bioinformatics.sdstate.edu/go/).
Sequencing files are stored in the BioProject PRJNA1021673 of the NCBI repository.

Transposon-sequencing and analysis
We used a pool of ~10 5 transposon-insertion mutants, generated by Tn5-based transposon T8 (ISlacZhah-tc) insertion in the P. aeruginosa strain PAO1 [28], and 20 μl of frozen Tn mutants (approximately containing 10 8 cells) was thawed and inoculated in 50 ml media or 50 ml media with 10 8 C. albicans cells in a 250-ml flask, to establish monoculture and co-culture conditions, each with 3 biological replicates.To screen the fitness of Tn mutants effectively, cells were grown at 30 ˚C with shaking at 210 rpm for 10 h (approximately 10 generations) and collected for genomic DNA extraction using the DNAeasy Blood & Tissue kit (Qiagen).DNA samples were prepared for transposon sequencing following the previously published TdT/ two-step PCR method [90].In short, 2 μg DNA was end-repaired by NEB end-repair reaction and subsequently C-tailed in the TdT tailing reaction.Two PCR reactions were used to enrich DNA fragments spanning the junction of transposon insertion.PCR-1 was performed using a transposon-specific primer and a polyG primer (olj376) on 0.5 to 1 μg DNA; at least 2 reactions which were pooled in the following purification steps.For PCR-2, a mixture of Tn8 primers combined with 3N, 5N, 7N, and 9N random bases were used to increase the diversity of Tn-seq libraries.The size of library DNA was examined by 4200 High Sensitivity DNA TapeStation System (Agilent) and quantified using qPCR with standard Illumina primers (Kapa Biosystems).Libraries were pooled and sequenced commercially using an Illumina NovaSeq or NextSeq sequencer (Novogene).For each sample, at least 25 million pair-end 150 bp sequencing reads were generated after which, we proceeded for downstream analysis.Sequencing files are stored in the BioProject PRJNA1021673 of the NCBI repository.The analysis scripts used in this study are adapted from previously published protocols and are available in the GitHub repository (https://github.com/PhoebeHsieh-yuying/P.-aeruginosa_Tnseq_paper) and the Zenodo repository (DOI: 10.5281/zenodo.11404260).Briefly, sequencing reads were groomed by FastQC (Barbaham Bioinformatics, Babraham Institute), filtered to keep those with the correct Tn8 sequence (TATAAGAGTCAG) near the 5 0 end using Cutadapt [91] and mapped to the P. aeruginosa PAO1 genome (GCF_00006765.1) with Bowtie2 [92] using the Tnseq2.shscript.The output table with transposon insertion site assignments of each sample was concatenated and annotated with known gene features of P. aeruginosa using a custom R script, Tnseq_reformat.R.Then, DESeq2 [89] statistical package in R was used to identify genes with significant changes in Tn insertion between co-culture and monoculture.A log 2 fold change cutoff of 1 and an FDR of 0.05 were used as the criteria for significance.

Experimental evolution of colistin survival
Eight independent colonies of a WT P. aeruginosa strain PAO1 were used as ancestral strains for the experimental evolution experiment.Each ancestral clone was inoculated and grown in BHI at 37 ˚C to 10 9 cells/ml.At the beginning of the experiment, approximately 10 7 cells of each ancestral population were transferred either into 1 ml BHI with colistin or into 1 ml BHI with colistin and 10 6 C. albicans cells to select colistin resistance.Each day, populations were passaged at a ratio of 1:200 in the former condition, while co-cultured populations were passaged at 1:50 in colistin-containing BHI to maintain comparable population size between both conditions.The concentration of colistin was started from 1.5 μg/ml, gradually increased 2-fold every week or every 2 weeks until it reached 192 μg/ml at the end of the 90-day evolution experiment.As controls for adaptation to BHI only or co-culture with C. albicans, the same ancestral populations were passaged at a ratio of 1:200 into 1 ml BHI or 1 ml BHI with the same amount of C. albicans cells, but without colistin.After every 7 days, 1 ml evolving cultures were mixed with 500 μl 80% glycerol and frozen at −80 ˚C.On day 90, we expected that populations evolved in colistin-containing BHI with C. albicans had been passaged for approximately 600 generations, while the rest of the evolved populations had been passaged for approximately 700 generations.

Whole-genome sequencing and analysis
We sequenced 2 ancestral populations and all 8 evolved populations per treatment.We revived each population from a freezer stock in the growth condition under which they evolved and grew for 24 h.For co-culture evolved populations, we treated them with nystatin to remove fungal cells, and 3 ml bacterial culture was collected for DNA extraction using the DNAeasy Blood & Tissue kit (Qiagen).Sequencing libraries were made and sequenced commercially by Illumina sequencers in SeqCenter (https://www.seqcenter.com/).Variant calling was done using the breseq [93] software v0.37.1 and the P. aeruginosa PAO1 genome (GCF_00006765.1) was used as the reference genome.The average depth of sequencing for populations was 222.5 fold (+/− 32.7) and the average genome coverage was 98.8 fold (+/− 0.04).Variants at >95% in each population were filtered for mutation calling.Mutations were manually curated by identifying unique variants in each evolved population compared to the ancestor.
Sequencing files are stored in the BioProject PRJNA1021673 of the NCBI repository.

Mg 2+ measurements in media
Mg 2+ concentrations were measured using the Magnesium Assay kit (Sigma-Aldrich) according to the protocol provided.Absorbances at OD 450 after the enzymatic reaction were compared to a standard curve of Mg 2+ concentration to determine the absolute concentration in media.

Fig 1 .
Fig 1. P. aeruginosa fitness is suppressed by C. albicans-mediated Mg 2+ sequestration.(A) A pool of P. aeruginosa Tn mutants was grown in BHI media only or in co-culture with C. albicans in BHI.Quantification of Tn mutant frequencies (see Methods) revealed genes whose loss either impaired P. aeruginosa fitness (blue; fungal-defense genes) or conferred a fitness advantage (red; dispensable genes) in co-culture with C. albicans.(B) Tn-seq volcano plot (see Methods) shows P. aeruginosa genes important for defense or dispensable in co-culture with C. albicans.X-axis indicates fold change, while Y-axis indicates p-value after correcting for multiple testing.We used log 2 fold change > 2 and adjusted p-value < 0.1 as statistical cutoffs.(C) Candidate defense genes identified by Tn-seq.A full list of these genes is available in S2 Table.(D)Fitness of P. aeruginosa single deletion ΔPA4824 or ΔmgtA mutants was impaired relative to WT P. aeruginosa in co-culture with C. albicans (orange) but not monoculture in BHI media (blue).The fitness of double deletion ΔPA4824 ΔmgtA mutant was even further impaired than single deletion mutants.CFUs of P. aeruginosa were measured by serially diluting cultures on LB+Nystatin.(E) (left panel) Intracellular Mg 2+ in P. aeruginosa is measured using an RNA sensor.When the intracellular Mg 2+ level is sufficient, mgtA 5 0 UTR forms a secondary structure that blocks downstream transcription.In limiting Mg 2+ , this structure is resolved, and transcription of a β-galactosidase reporter is restored (right panel).Intracellular Mg 2+ levels, measured in β-galactosidase units, of single deletion mutants lacking PA4824 or mgtA were lower than the WT stain and even lower in a double deletion ΔPA4824 ΔmgtA mutant in C. albicans-spent BHI media (orange), but not BHI media alone (blue).(F) Co-culture-specific fitness of the P. aeruginosa single or double deletion mutants relative to WT strains was restored by Mg 2+ supplementation (10 mM) in BHI in co-culture (orange).Mean ± std of 3 biological replicates is shown in panels D-F.(** p < 0.01 Dunnett's one-way ANOVA test used; n.s.indicates not significant).The data underlying Fig 1D-1F can be found in S6 Data.Fig 1A and 1E are created with Biorender.com.BHI, brain heart infusion; CFU, colony-forming unit; WT, wild-type.
Fig 1. P. aeruginosa fitness is suppressed by C. albicans-mediated Mg 2+ sequestration.(A) A pool of P. aeruginosa Tn mutants was grown in BHI media only or in co-culture with C. albicans in BHI.Quantification of Tn mutant frequencies (see Methods) revealed genes whose loss either impaired P. aeruginosa fitness (blue; fungal-defense genes) or conferred a fitness advantage (red; dispensable genes) in co-culture with C. albicans.(B) Tn-seq volcano plot (see Methods) shows P. aeruginosa genes important for defense or dispensable in co-culture with C. albicans.X-axis indicates fold change, while Y-axis indicates p-value after correcting for multiple testing.We used log 2 fold change > 2 and adjusted p-value < 0.1 as statistical cutoffs.(C) Candidate defense genes identified by Tn-seq.A full list of these genes is available in S2 Table.(D)Fitness of P. aeruginosa single deletion ΔPA4824 or ΔmgtA mutants was impaired relative to WT P. aeruginosa in co-culture with C. albicans (orange) but not monoculture in BHI media (blue).The fitness of double deletion ΔPA4824 ΔmgtA mutant was even further impaired than single deletion mutants.CFUs of P. aeruginosa were measured by serially diluting cultures on LB+Nystatin.(E) (left panel) Intracellular Mg 2+ in P. aeruginosa is measured using an RNA sensor.When the intracellular Mg 2+ level is sufficient, mgtA 5 0 UTR forms a secondary structure that blocks downstream transcription.In limiting Mg 2+ , this structure is resolved, and transcription of a β-galactosidase reporter is restored (right panel).Intracellular Mg 2+ levels, measured in β-galactosidase units, of single deletion mutants lacking PA4824 or mgtA were lower than the WT stain and even lower in a double deletion ΔPA4824 ΔmgtA mutant in C. albicans-spent BHI media (orange), but not BHI media alone (blue).(F) Co-culture-specific fitness of the P. aeruginosa single or double deletion mutants relative to WT strains was restored by Mg 2+ supplementation (10 mM) in BHI in co-culture (orange).Mean ± std of 3 biological replicates is shown in panels D-F.(** p < 0.01 Dunnett's one-way ANOVA test used; n.s.indicates not significant).The data underlying Fig 1D-1F can be found in S6 Data.Fig 1A and 1E are created with Biorender.com.BHI, brain heart infusion; CFU, colony-forming unit; WT, wild-type.https://doi.org/10.1371/journal.pbio.3002694.g001

Fig 2 .
Fig 2. Mg 2+ competition is widespread between multiple fungal and gram-negative bacterial species.(A) The fitness of the E. coli ΔmgtA mutant was impaired relative to the WT strain in co-culture with C. albicans (orange) but not in monoculture (blue) in BHI media.The fitness of the ΔmgtA strain was restored in BHI media supplemented with 10 mM Mg 2+ .(B) The fitness of the double deletion ΔmgtA ΔmgtB mutant S. Typhimurium strain was impaired relative to WT in co-culture with C. albicans (orange) but not in monoculture in BHI media (blue).(C) The fitness of the ΔmgtA mutant P. aeruginosa strain (green) was impaired relative to WT (magenta) in BHI in co-culture with fungal species: C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and S. cerevisiae.(D) Mg 2+ supplementation restored the fitness of the ΔmgtA mutant P. aeruginosa strain (green) in co-culture with fungal species in Fig 2C.(E) The fitness of the P. aeruginosa ΔmgtA mutant (green) does not significantly differ from WT (magenta) in co-culture with gram-positive S. aureus.(F) The fitness of the P. aeruginosa ΔmgtA mutant (green) was similar to WT (magenta) in co-culture with gram-negative bacteria, such as B. multivorans, E. coli, or S. Typhimurium.(G) The functions and locations of proposed Mg 2+ transporters in S. cerevisiae are illustrated with Biorender.com(top panel).Alr2 localizes to the plasma membrane, Mrs2 and Mme1 to the mitochondria, and Mnr2 to the vacuole.The fitness of the P. aeruginosa ΔmgtA mutant (black) is not

Fig 3 .
Fig 3. C. albicans-mediated Mg 2+ sequestration protects gram-negative bacteria from colistin.(A) Mg 2+ binds to negatively charged lipid A of LPS on the outer membrane of gram-negative bacteria.In high Mg 2+ conditions, colistin (and other polymyxin antibiotics) targets Mg 2+ -bound LPS to disrupt cell membranes of gram-negative bacteria, leading to bacterial cell death.Low Mg 2+ conditions activate two-component signaling pathways, PmrAB and PhoPQ, that modify LPS (PEtN and L-Ara4N), making bacteria resistant to colistin.(B) Schematic of the colistin survival assay in C. albicans-spent media.P. aeruginosa strains were grown to stationary phase in BHI, C. albicans-spent BHI, or C. albicans-spent BHI supplemented with 10 mM Mg2+ , and then passaged until exponential phase, when they were treated with 25 μg/ml colistin.Bacterial survival is shown as the percentage of viable cells after the colistin treatment.(C) WT P. aeruginosa strains were highly susceptible to colistin treatment in BHI media.However, they could survive colistin treatment in C. albicans-spent BHI media but not in spent BHI media supplemented with 10 mM Mg2+ .In contrast to WT strains, pmrB or phoP Tn-mutants of P. aeruginosa (which cannot modify LPS) remained highly susceptible to colistin (G) Like in (F), co-cultures of S. Typhimurium with C. albicans (orange) were significantly more resistant than monocultures of S. Typhimurium alone over a range of colistin concentrations.Mean ± std of 3 biological replicates is shown in panels C-D and F-G.(**p < 0.01, Dunnett's one-way ANOVA test and unpaired two-tailed Student's t test were used in panel C-D and F-G separately).The data underlying this Fig 3C-3D and 3F-3G can be found in S13 Data.Fig 3A, 3B, and 3E are created with Biorender.com.BHI, brain heart infusion; LPS, lipopolysaccharide; WT, wild-type.https://doi.org/10.1371/journal.pbio.3002694.g003

Fig 4 .
Fig 4. Long-term coexistence with C. albicans alters the evolution of P. aeruginosa colistin resistance.(A) Schematic of the evolution experiment in coculture, created with Biorender.com.Eight independent WT P. aeruginosa populations were passaged daily in BHI with colistin or in BHI with both C. albicans and colistin.Colistin levels were gradually increased from 1.5 μg/ml to 192 μg/ml (128× clinical MIC) over 90 days (see Methods).(B) After evolution, 8 monoculture replicate populations (blue) were not significantly different from 8 co-culture populations (orange) for resistance to 192 μg/ml colistin (co-culture populations were treated with colistin in the presence of co-evolving fungal cells).For each population, the average survival rate of 4 replicates is shown (n.s.indicates not significant, Mann-Whitney U test).(C) P. aeruginosa cells from co-cultured-evolved populations (orange circle) lost colistin resistance upon removal of C. albicans with antifungal nystatin treatment (gray circle) or upon Mg 2+ supplementation (yellow triangle).Mean ± std of 4 biological replicates is shown (* p < 0.05, ** p < 0.01, Mann-Whitney U test).(D) Genome sequencing revealed that P. aeruginosa strains that evolved enhanced colistin survival either in monoculture or in co-had different numbers of fixed mutations per population (** p < 0.01, Mann-Whitney U test) due to recurrent hypermutator mutations in monoculture populations.(E) Monoculture-and co-culture-evolved populations acquired enhanced colistin survival via a distinct spectrum of putative adaptive mutations.Columns indicate each individual replicate monoculture (blue) or co-culture (orange)-evolved population.Rows indicate genes that acquire mutations, grouped by cellular processes.We list all genes with fixed mutations (gray) in 5 or more replicate populations in either condition (* indicates a mutation in the promoter region).The only genes common between monoculture and co-culture-adapted populations are colS, previously known for colistin resistance, and PA3969 (indicated with a #), which is not unique to colistin-treated populations.A more comprehensive list of all fixed mutations is presented in S3 and S4 Tables.The data underlying this Fig 4B-4D can be found in S19 Data.BHI, brain heart infusion; WT, wild-type.https://doi.org/10.1371/journal.pbio.3002694.g004

Fig 5 .
Fig 5. Mg 2+ sequestration by fungi suppresses bacterial fitness but profoundly impacts the acquisition of colistin resistance.We present a model to summarize our findings.C. albicans sequesters essential Mg 2+ ions from P. aeruginosa.In turn, the bacterium competes for Mg 2+ ions using the Mg 2+ transporter, MgtA.Fungal-mediated Mg 2+ sequestration alters LPS modification on the bacterial outer membranes and enhances bacterial resistance to colistin.In co-culture with C. albicans, P. aeruginosa evolves non-canonical fungal-dependent colistin resistance, which can be disrupted by fungal removal or Mg 2+ supplementation.This figure is created with Biorender.com.LPS, lipopolysaccharide.https://doi.org/10.1371/journal.pbio.3002694.g005

S4Fig.
Competitive fitness assay verifies the fitness effect of PA4824 or mgtA mutants in co-culture.(A) Schematic of the fluorescence-based competitive fitness assay that mimics bulk selection in Tn-seq (see Methods).(B) Relative fitness of PA4824 or mgtA mutants to the WT P. aeruginosa strain was impaired in co-culture conditions.Relative fitness in BHI media only is indicated in blue, and relative fitness in co-culture with C. albicans is indicated in orange.Mean ± std of 3 biological replicates is shown (** p < 0.01, unpaired two-tailed Student's t test used).(C) A WT copy of mgtA restored the fitness of ΔmgtA mutant in co-culture.Mean ± std of 4 biological replicates is shown (** p < 0.01, n.s.indicates not significant, Dunnett's oneway ANOVA test was used).(D) The fitness of ΔPA4824 or ΔmgtA mutant was significantly restored by arabinose-induced expression of PA4824 or mgtA.ΔPA4824 was transformed with pJN105-PA4824 and ΔmgtA was transformed with pJN105-mgtA.Deletion mutants with pJN105 (vector only) were used as controls.Mean ± std of 4 biological replicates is shown (** p < 0.01, unpaired two-tailed Student's t test was used).The data underlying S4B-S4D Fig can be found in S5 Data.(EPS) S5 Fig. Verification of the Mg 2+ genetic sensor in P. aeruginosa.The WT P. aeruginosa strain carrying the Mg 2+ genetic sensor (pBBR-P lac1-6 -mgtA leader -lacZ) was cultured in minimal medium with 10 μm or 10 mM Mg 2+ to log phase, mimicking low or high Mg 2+ conditions.Relative concentrations of intracellular Mg 2+ were determined by the measurements of β-galactosidase activity (see Methods).Mean ± std of 3 biological replicates is shown (** p < 0.01, unpaired two-tailed Student's t test used).The data underlying this figure can be found in S7 Data.(EPS) S6 Fig. Alphafold structural prediction suggests PA4824 may function as a Mg 2+ transporter.(A) An Alphafold-predicted structure of PA4824.Confidence in structural prediction is shown by color, where dark and light blue indicate high confidence.(B) and (C) show the top-down view of the core of PA4824, displayed by Chimerax.In (B), residues are labeled by their hydrophobicity (green: hydrophilic residues, yellow: hydrophobic residues).In (C), residues are labeled by their electrostatic features (red: negatively charged residues, blue: positively charged residues).(EPS) S7 Fig. Two other P. aeruginosa Mg 2+ transporters, CorA and MgtE, are not involved in Mg 2+ competition with C. albicans.(A) Expression levels of mgtE and corA were unchanged in response to C. albicans presence.RNA-seq results of mgtE, corA, and mgtA across 4 experimental conditions (BHI only, co-culture in BHI, BHI with supplemental Mg 2+ (10 mM), and co-culture in BHI supplemental Mg 2+ (10 mM)) are shown in z-score.The log 2 fold change of each gene in co-culture versus monoculture in BHI is shown on the right.(B) corA or mgtE Tn mutants did not confer fitness cost in co-culture with C. albicans, whereas ΔmgtA mutant did.Fitness of P. aeruginosa strains in BHI only or co-culture conditions are shown in blue or orange, respectively.Mean ± std of 4 biological replicates is shown (** p < 0.01, * p < 0.05, and n.s.indicates not significant.Dunnett's one-way ANOVA test was used).(C) corA or mgtE mutants did not show reduced intracellular Mg 2+ levels in C. albicans spent BHI, whereas ΔmgtA mutants did.The same experimental setup in Fig 1E was used for this assay.Mean ± std of 3 biological replicates is shown (** p < 0.01, Dunnett's one-way ANOVA test was used).The data underlying S7A-S7C Fig can be found in S8 Data.(EPS) S8 Fig. Mg 2+ supplementation rescues the fitness effects of several candidate genes, without altering their expression.(A) Tn-seq volcano plot in Mg 2+ -repleted conditions (see Methods) shows the fitness effects of most of the candidate defense and dispensable genes (in black) were restored (compare to Fig 1B).X-axis indicates fold change, while Y-axis indicates p-value after correcting for multiple testing.We used log 2 fold change >2 and adjusted p-value < 0.1 as statistical cutoffs.PA4824, PA4825, and PA4826 are labeled in yellow.Under this condition, only 1 gene was found to be dispensable (in red, PA2854), and 2 genes were found to be essential (in blue, PA5484 and PA4029).(B) Mg 2+ supplementation only altered the expression of PA4824, PA4825, and PA4826 in co-culture, but not the other candidate genes.Heatmap shows the expression of 18 dispensable genes and 8 fungal-defense genes in co-culture versus monoculture, with or without Mg 2+ supplementation.The data underlying S8A and S8B Fig can be found in S9 Data.(EPS) S9 Fig. Mg 2+ -sequestration by fungi depends on environmental Mg 2+ levels and primarily determines bacterial fitness in co-culture.(A-C) Fitness of P. aeruginosa ΔmgtA mutant was measured in co-culture with C. albicans in TSB (A), SCFM (B), or YPD (C).

Differentially expressed P. aeruginosa genes in co-culture relative to monoculture conditions.
During co-culture in BHI for 42 h, P. aeruginosa fitness was significantly impaired relative to monoculture, by nearly 100-fold at 20 h.In contrast, C. albicans fitness was similar in monoculture versus co-culture (see Methods).The fitness of C. albicans or P. aeruginosa is shown as colony-forming units monoculture versus co-culture for the first 8 h but began to significantly diverge by the 10-h time point.Mean ± std of 3 biological replicates is shown (** p < 0.01 and n.s.indicates not significant.Unpaired two-tailed Student's t test was used, relative to monoculture at each time point).The data underlying this figure can be found in S2 Data.Gene Ontology analysis of genes up-regulated (A) or down-regulated (B) in coculture compared to monoculture is shown.Up-regulated genes were enriched for iron uptake and RNA polymerase sigma factor 70, whereas down-regulated genes were enriched for bacterial secretion, co-factor biosynthesis, and energy generation.X-axis indicates fold enrichment of genes of interest, and Y-axis indicates GO categories, which are ordered and color-coded by the inverse of their false discovery rate.The number of genes of interest in each category is labeled with the size of circles.(C) RNA-seq volcano plot (see Methods) shows P. aeruginosa genes that were differentially expressed in co-culture versus monoculture conditions.X-axis indicates fold change, while Y-axis indicates p-value after correcting for multiple testing.|log 2 fold change| > 2 and adjusted p < 0.05 was used as statistical cutoffs.Up-regulated genes are labeled in red, while down-regulated genes are in blue.The data underlying this figure can be found in S3 Data.(EPS) S1 Fig. P. aeruginosa fitness is suppressed by C. albicans in BHI.S2 Fig. P. aeruginosa fitness remains unchanged during the first 8 h in co-culture with C.albicans.The WT P. aeruginosa strain was grown in BHI media only or in co-culture with C. albicans (see Methods).The fitness of P. aeruginosa was examined every 2 h in BHI (blue) and co-culture (orange) by measuring CFU per ml.P. aeruginosa fitness was not significantly different in The ΔmgtA mutant had impaired fitness in TSB (like in BHI media), but not SCFM or YPD media.Bacterial fitness in media only or in co-culture is shown in blue or orange, respectively.Mean ± std of 4 biological replicates is shown (** p < 0.01 and n.s.indicates not significant.Unpaired two-tailed Student's t test was used).(D) BHI and TSB exhibited lower Mg 2+ levels compared to YPD and SCFM, both in the fresh media and in C. albicans spent media.Mean ± std of 3 biological replicates is shown.(E) Intracellular Mg 2+ levels in P. aeruginosa were lower in the fungal spent media from BHI and TSB but not in fungal spent media from SCFM or YPD.Mean ± std of 4 biological replicates is shown.(F) Fitness of the P. aeruginosa ΔmgtA mutant in co-culture was rescued by adding more than 0.3 mM Mg 2+ , leading to a total of 0.45 mM Mg 2+ in BHI.(G) Supplementation of other cations (Zn 2+ , Cu 2+ , Mo 6+ , or Mn 2+ ) in co-culture did not restore the fitness of P. aeruginosa ΔmgtA mutant.Mean ± std of 4 biological replicates is shown (** p < 0.01; unpaired two-tailed Student's t test was used).(H) Fitness of P. aeruginosa ΔmgtA mutant in co-culture could only be rescued by supplementation of 10 mM Ca 2+ .Mean ± std of 4 biological replicates is shown (n.s.indicates not significant.Unpaired two-tailed Student's t