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Widespread fungal–bacterial competition for magnesium lowers bacterial susceptibility to polymyxin antibiotics [1]

['Yu-Ying Phoebe Hsieh', 'Division Of Basic Sciences', 'Howard Hughes Medical Institute', 'Fred Hutchinson Cancer Center', 'Seattle', 'Washington', 'United States Of America', 'Wanting Sun', 'Janet M. Young', 'Robin Cheung']

Date: 2024-06 

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 Mg 2 + sequestration, P. aeruginosa expresses the Mg 2 + transporter MgtA when Mg 2 + levels are low. Thus, loss of MgtA specifically impairs P. aeruginosa in co-culture with C. albicans, but fitness can be restored by supplementing Mg 2 + . Using a panel of fungi and bacteria, we show that Mg 2 + sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg 2 + 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.

Funding: This work was supported by a postdoctoral fellowship from the Cystic Fibrosis Foundation (HSIEH21F0 to Y-YPH) and grants from the National Institutes of Health (R01GM125714, R35GM152107 to AAD; R01AI127548 to DAH), from the Burroughs Wellcome Fund (1012253 to AAD), and from the Howard Hughes Medical Institute (Investigator award to HSM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting information files. Sequencing files are stored in the BioProject PRJNA1021673 of the NCBI repository. Analysis scripts are available in the GitHub repository ( https://github.com/PhoebeHsieh-yuying/P.-aeruginosa_Tnseq_paper ) and the Zenodo repository (DOI: 10.5281/zenodo.11404260 ). Strains and plasmids are available upon request.

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.

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.

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 – 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 ].

Results

To identify strategies used by fungi to antagonize bacteria, we first investigated whether co-culture 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 [28–30] to conduct a genome-wide fitness screen in bacteria. We cultured a pool of 105 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.

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TIFF original image Download: Fig 1. P. aeruginosa fitness is suppressed by C. albicans-mediated Mg2+ 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 Mg2+ in P. aeruginosa is measured using an RNA sensor. When the intracellular Mg2+ level is sufficient, mgtA 5′UTR forms a secondary structure that blocks downstream transcription. In limiting Mg2+, this structure is resolved, and transcription of a β-galactosidase reporter is restored (right panel). Intracellular Mg2+ 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 Mg2+ 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

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