Experimental evolution of a fungal pathogen into a gut symbiont

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Science  02 Nov 2018:
Vol. 362, Issue 6414, pp. 589-595
DOI: 10.1126/science.aat0537

Gut microbiota selects fungi

Fungi, such as Candida albicans, are found in the mammalian gut, but we know little about what they are doing there. Tso et al. put C. albicans under evolutionary pressure by serial passage in mice that were treated with antibiotics and were thus lacking gut bacteria (see the Perspective by d'Enfert). Passage accelerated fungal mutation, especially around the FLO8 gene, resulting in low-virulence phenotypes unable to form hyphae. Nevertheless, these phenotypes stimulated proinflammatory cytokines and conferred transient cross-protection against several other gut inhabitants. However, if an intact microbiota was present, only the virulent hyphal forms persisted.

Science, this issue p. 589; see also p. 523


Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen Candida albicans in the mouse gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of “trained immunity.” Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus.

Symbiotic relationships are ubiquitous in nature and vary on a continuum from parasitism to mutualism (1). Evolution plays a critical role in the establishment of these interactions and in moving them along the parasite-mutualist axis. In the case of host-microbe interactions, the mode of host-to-host transmission dictates whether the microbe will evolve toward higher pathogenicity, commensalism, or mutualism (2). Experimental evolution via serial passaging of pathogens in a new host is a powerful strategy to study these dynamic changes in real time. For instance, passaging parasites in new hosts via the systemic infection route most often selects for increased virulence against the new host (3). However, experimental systems to study the evolutionary emergence of mutualistic animal-microbe interactions are lacking (4).

The mammalian gastrointestinal (GI) tract harbors a large and diverse microbial community, whose members interact with the host primarily in commensal or mutualistic ways (5). This raises the question of how the mammalian host establishes these neutral or beneficial interactions while purging potential pathogens. We hereby hypothesize that the gut environment selects microbes on the basis of their pathogenicity and moves them along the parasitism-mutualism axis via evolutionary processes.

To investigate how the gut environment shapes the evolution of a microbe, we developed an experimental system, based on long-term GI colonization of antibiotic-treated mice by the fungus Candida albicans coupled with serial fecal transplants from colonized to naïve hosts. Several variations of the protocol were tested (Fig. 1A), and all fecal transplants resulted in successful colonization of the recipient animals. Clonal isolates harvested after 8 or 10 weekly serial passages (w8 or w10 strains), but not after a 1-week passage (w1 strains), showed a significantly increased intra-GI competitive fitness (Fig. 1B), which was at least as high as that of strains deficient of the enhanced filamentous growth 1 (EFG1) gene (efg1−/− strains), which were previously known to be hyperfit in the antibiotic-treated mouse gut (68). Overall, these results demonstrate that a microbe can be experimentally induced to increase its competitive fitness in the gut of an unnatural host by means of adaptive evolution.

Fig. 1 Adaptive evolution of C. albicans in the mouse gut selects for hyphal-defective variants.

(A) Schematic overview of the different evolution protocols. Individual strains are named according to line (L), week (w), and colony (c). Some specific strains are additionally identified by a shorter name (e.g., W2N or R24). (B) Similar to the efg1−/− (efg1/efg1) mutant, 10-week (w10) and 8-week (w8) C. albicans strains evolved in antibiotic-treated mice achieved increased competitive fitness in the mouse GI tract compared with WT (SC5314) and 1-week (w1) gut-evolved strains (W1 and R1). However, after 5 weeks of evolution in antibiotic-free infants, C. albicans strains failed to increase their competitive fitness when compared with strains evolved in antibiotic-treated infants. Data are means ± SD. n = 4 to 10 mice per group. Mann-Whitney test: **P < 0.01, ***P < 0.001, and ****P < 0.0001. (C) Whole-stool populations from evolution lines L44 to L58 were plated on Spider agar and scored for smooth (indicative of yeast) and wrinkled (indicative of filamentous) colonies. Smooth colonies eventually appeared and often dominated the stool populations in all antibiotic-treated (Infant+), but not antibiotic-free (Infant−), lines. (D) Individual strains (clonal isolates) of each evolution line were scored for hyphal formation in response to serum. Percentages of hyphal-proficient strains were significantly lower at the end (week 8 or 10, depending on the protocol) than after 1 week of the evolution experiment. Gray indicates no data. (E) Representative images indicating that gut-evolved C. albicans strains are defective in hyphal formation in response to in vitro stimuli. Black scale bar, 200 μm; red scale bar, 20 μm.

Host adaptive immunity appeared to play little or no role in this adaptation process, because all results presented herein were qualitatively similar whether the evolution experiments were performed in wild-type (WT) or Recombination activating 1–deficient (Rag1−/−) mice, which lack functional T and B cells. To determine if antibiotic treatment played any role in this evolutionary process, we repeated the above evolution experiments in untreated mice. Without the use of antibiotics, however, and consistent with current knowledge (9), we were unable to establish a long-term colonization in the adult animals’ guts, and the experiment had to be aborted after 3 weeks (Fig. 1A). We thus resorted to an alternative model involving neonatal mice, which have long been known to allow C. albicans GI colonization even in the absence of antibiotics (10). Specifically, we intragastrically inoculated 2-week-old infant mice with the same ancestral C. albicans used for the previous evolution experiments, with seven evolution lines maintained on oral antibiotics and eight lines on control water (Fig. 1A). Notably, individual colonies isolated after 5 weeks of evolution displayed a significantly increased GI competitive fitness in infant mice only if they had been evolved in antibiotic-treated, but not untreated, mice (Fig. 1B), suggesting that the presence of an intact bacterial microbiota limits the adaptation of C. albicans in the mouse GI tract.

Because C. albicans strains that are hyperfit in the antibiotic-treated or germ-free mouse gut are typically deficient in hyphal morphogenesis (6, 8, 11), we next examined cellular morphologies and noted that over the course of 10 weeks of evolution in infant mice, all C. albicans populations progressively lost their ability to respond to hyphal-inducing stimuli, but only if they had been evolved in the presence of antibiotics (Fig. 1C). Screening several independent clones across all evolution lines, we confirmed that in the presence of antibiotics, virtually all C. albicans cells lost their ability to form true hyphae by the end of each experiment (Fig. 1, D and E). Importantly, in vitro daily serial passaging of C. albicans for 5 weeks in the presence of antibiotics failed to yield such phenotypes (fig. S1). Though experimental evolution in germ-free mice would be the ideal model to definitively prove this point, these data strongly suggest that it was the absence of microbiota and not antibiotics per se that selected for hyphal-defective strains in our in vivo evolution experiments. Finally, nonfilamentous w10 strains and efg1−/− cells, although being hyperfit in the antibiotic-treated gut, actually colonized the GI tract of untreated mice less efficiently than WT cells (fig. S2). Together, these results indicate that, although the hyphal morphogenesis program is required for competition of C. albicans with resident gut bacteria, it otherwise represents a fitness burden and is thus rapidly lost when the gut microbiota are absent or chronically perturbed by long-term antibiotic treatment.

In search of the genetic basis underlying these evolved phenotypes, we performed high-depth, high-coverage whole-genome sequencing on four ancestral C. albicans strains used as inocula for the evolution experiments, four w1 strains, and 28 w5, w8, or w10 strains selected from 16 independent evolution lines across three different serial passaging protocols (table S1). A total of 34 verified open-reading frames were identified as carrying ≥1 de novo nonsynonymous mutation in ≥1 evolved w5, w8, or w10 strain (Fig. 2A and table S2). On the basis of Gene Ontology enrichment analysis, 20 of these genes were related to filamentous growth (P = 5.5 × 10−10, hypergeometric test, Bonferroni correction), 10 encoded proteins located in the cell wall (P = 3.5 × 10−7, hypergeometric test, Bonferroni correction), and seven functioned as transcription factors (TFs) (P = 2.2 × 10−3, hypergeometric test, Bonferroni correction). Specifically, six genes (e.g., EFG1) encoded TFs involved in hyphal gene expression regulation (table S3).

Fig. 2 Recurrent mutations in TFs regulating filamentous growth underlie increased competitive fitness in the mouse gut.

(A) Clustering of gut-evolved C. albicans strains based on mutational pattern across 34 verified open-reading frames carrying de novo, nonsynonymous substitutions (DNSs) reveals recurrent mutations in FLO8 and other TFs required for filamentous growth. Genes are ordered on the basis of chromosomal location. (B) Convergent acquisition of frameshift (fs) or nonsense (*) mutations in the FLO8 gene across multiple independent evolution experiments, most often in homozygosity. (C) FLO8 and EFG1 act downstream of the cyclic adenosine 3′,5′-monophosphate (cAMP) pathway to regulate the expression of hypha-specific genes in response to environmental stimuli, such as serum (28). (D) The flo8−/− (flo8/flo8) mutant and two w10 evolved strains (W2N and L11.w10.c3) harboring FLO8 loss-of-function mutations were unable to filament in hyphal-inducing media. By contrast, the flo8+/ (flo8/FLO8) mutant and the w10 evolved strains, in which one functional FLO8 allele had been restored (flo8R/−), but not control strains transformed only with the selection marker (flo8S/−), were able to form hyphae. Black scale bar, 200 μm; red scale bar, 20 μm. (E) Although flo8−/− cells were significantly fitter than flo8+/ cells in the mouse GI tract, restoration of one FLO8 allele to its ancestral sequence significantly decreased the GI fitness of strains W2N and L11.w10.c3. Data are means ± SD. n = 5 mice per group. Mann-Whitney test: **P < 0.01 and ***P < 0.001.

With 22 of 28 sequenced evolved strains (or 11 out of 16 evolution lines) carrying ≥1 de novo mutation in FLO8, which encodes a TF required for hyphal development (Fig. 2C) (12), this gene was the most frequently mutated (Fig. 2A). Interestingly, all identified FLO8 mutations lead to premature stop codons or frame shifts (Fig. 2B), implicating these as loss-of-function mutations. In accordance, many stop codons were located near amino acid position 654, where truncations were shown to result in null phenotypes (13). To test if FLO8 inactivation is sufficient to increase the fitness of C. albicans in the mouse GI tract, we generated heterozygous (flo8+/−) and homozygous (flo8−/−) FLO8-deletion strains not previously exposed to the mouse gut environment. Consistent with our predictions, flo8−/−, but not flo8+/−, cells were nonfilamentous (Fig. 2D) and outcompeted WT C. albicans as efficiently as the efg1−/− strain or any of the gut-evolved C. albicans strains (Fig. 2E). To test if the single-point mutations identified in the w10 strains were indeed required for increased competitive fitness, we restored one FLO8 allele back to its ancestral sequence in strains w7 and L11.w10.c3 and observed reversion of filamentation (Fig. 2D) and a significant loss of intra-GI tract fitness in both strains (Fig. 2E). Further demonstrating these point mutations to be fully recessive and not dominant-negative, ectopic expression of a WT FLO8 under its own promoter fully restored filamentation in the tested w10 strains, whereas a mutated FLO8 had no effect when ectopically expressed in a WT strain (fig. S3). Consistent with this notion, 19 of the 22 strains carrying FLO8-inactivating mutations carried them in homozygosity (Fig. 2B). These results show that adaptive evolution in the antibiotic-treated mouse GI tract reproducibly selects for inactivating mutations in central transcriptional regulators of the hyphal morphogenesis program of C. albicans and, in particular, in FLO8, yielding hyphal-defective mutants with increased intra-GI tract fitness. Notably, a spectrum of hyphal-formation defects, in part associated with homozygous nonsense mutations in key hyphal morphogenesis TFs, such as EFG1, are reported to also occur in human clinical isolates of C. albicans (8).

Beyond single-nucleotide changes, C. albicans is known to frequently undergo large-scale genomic mutations, such as short- and long-range loss of heterozygosity (LOH) and segmental or whole-chromosome aneuploidy, especially when under stress and during infection (14, 15). In accordance, 24 of 28 w5, w8, and w10 evolved strains (15 of 16 evolution lines) underwent at least one LOH or aneuploidy event, with chromosome 6 (chr6) LOH (11 strains across six evolution lines) and chr7 trisomy (12 strains across 10 evolution lines) being the most frequently observed (fig. S4). Interestingly, FLO8 is located on chr6, and all observed chr6 LOH events occurred in strains with homozygous FLO8 mutations (Fig. 2A), suggesting that the LOH event followed the single-nucleotide change to allow expression of the recessive phenotype. Chr7 trisomy, instead, did not appear to contribute to the adaptive process, because an unevolved, independently obtained strain carrying this aneuploidy displayed neither filamentation defects nor an increased competitive fitness in the mouse GI tract (fig. S5).

As the hyphal morphogenesis program is a key virulence factor of C. albicans (16), we hypothesized that gut-evolved strains would be less damaging to host cells and be less pathogenic during infection. To test this, we first measured their cytotoxicity in cocultures with either murine macrophages or human colon epithelial cells. Similar to the efg1−/− or flo8−/− strain, we found that w10 strains induced significantly lower cell damage than WT or w1 strains (Fig. 3A). We next tested the virulence of gut-evolved strains in a mouse model of hematogenously disseminated candidiasis. As expected, using a lethal dose of 5 × 105 colony-forming units (CFUs) of WT C. albicans, we observed formation of hyphae in the kidneys of WT mice 2 days postinfection (dpi), as well as severe necrosis, moderate perivascular edema, and moderate diffuse pyogranulomatous renal capsulitis in most animals (Fig. 3B). Eventually, all mice infected with WT or w1 strains succumbed within 3 to 4 dpi (Fig. 3C). By contrast, at the same infection dose, w10 and flo8−/− strains strictly remained in the yeast form in kidneys at 2 dpi, whereas a few hyphae were observed with efg1−/− cells, and only mild to moderate necrosis was observed with the w10 strains, with only some animals displaying mild edema or capsulitis (Fig. 3B). Similar results were obtained when we infected Rag1−/− mice; mice infected by efg1−/−, flo8−/−, or w10 strains survived significantly longer than those infected by the WT or w1 strains (Fig. 3C). Again demonstrating a key role of the gut microbiome in this process, C. albicans strains evolved in the absence of, but not in the presence of, antibiotics retained their ability to kill mice (fig. S6). Notably, similar loss of virulence was observed both for w10 strains carrying and for those not carrying FLO8 mutations (Fig. 3, A and C). However, the two w10 strains that were “cured” of their FLO8-inactivating mutations displayed a significantly increased virulence compared to their w10 counterparts (fig. S7), indicating that those point mutations were required for the loss of virulence in those evolved strains. Hence, this experimental system, unlike serial passaging via the systemic infection route or in cell culture (1719), reproducibly yields C. albicans strains that are genetically locked in the yeast form and avirulent. And although FLO8 appears to be a mutational hotspot with a clear implication in this adaptation mechanism, convergent evolution toward similar phenotypes was also observed in strains lacking FLO8-inactivating mutations.

Fig. 3 Gut-evolved C. albicans strains are avirulent and protect gut-colonized hosts from systemic fungal infections.

(A) Similar to the efg1−/− and flo8−/− mutants, C. albicans w10 evolved strains with (filled circles) or without (empty circles) FLO8-inactivating mutations had reduced in vitro cytotoxicity against J774A.1 mouse macrophages and HT-29 human gut epithelial cells compared with WT (SC5314) and w1 evolved strains (W1 and R1). LDH, lactate dehydrogenase. Data are means ± SD. n = 3 independent experiments. Unpaired Welch’s t test: *P < 0.05, **P < 0.01, and ***P < 0.001, ****P < 0.0001. (B) One representative Periodic acid–Schiff–stained kidney sections of 3 to 5 mice per group infected with WT (SC5314), efg1/, flo8+/, flo8/−+, and C. albicans w10 evolved strains (W2N or R24). Black scale bar, 200 μm; red scale bar, 20 μm. Images on the right are magnifications of the boxed areas to the left. (C) Similar to the efg1−/− and flo8/ mutants, w10 evolved strains with (filled circles) or without (empty circles) FLO8-inactivating mutations are less virulent in WT and Rag1−/− mice compared with WT (SC5314) or w1 strains (W1 and R1). n = 5 to 10 mice per group. (D and E) Antibiotic-treated adults or antibiotic-free infants were colonized with either WT (SC5314) or a w10 strain (R24) and challenged systemically with either WT C. albicans (D) or A. fumigatus (E). In all tested cases, R24-colonized mice were qualitatively or quantitatively more protected from systemic fungal infections than SC5314-colonized mice. n = 8 to 25 mice per group. For (C) to (E), data were analyzed by log-rank test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Because gut colonization by commensal fungi protects hosts against infections (20), we next asked if hosts colonized by gut-evolved C. albicans strains carry a potential advantage over mice colonized with WT C. albicans. We first tested this hypothesis under conditions in which the evolved strains would express their increased competitive fitness, that is, in antibiotic-treated adult mice. Notably, animals colonized with the w10 strain R24 (which coincidentally does not harbor FLO8 mutations), but not with the ancestral WT strain SC5314, showed a significantly increased survival upon a systemic challenge with a fully virulent C. albicans strain (Fig. 3D). Under these conditions, both the evolved microbe and its new host benefit from their interaction.

We then also repeated the colonization in non-antibiotic-treated 2-week-old pups, which were then systemically challenged with the fully virulent strains at 6 weeks of age. Although R24 is more rapidly lost than WT in untreated pups (fig. S2), >30% of R24-colonized mice survived until 35 dpi, whereas all SC5314-colonized animals died by ~20 dpi (Fig. 3D). We also tested gut-colonized pups for protection against the distantly related fungus Aspergillus fumigatus. Whereas most uncolonized mice or SC5314-colonized mice succumbed ≤8 or ≤15 dpi, respectively, >20% of R24-colonized mice survived at least until 35 dpi (Fig. 3E). Therefore, GI colonization with C. albicans, and especially with a gut-evolved strain, at a young age protects hosts against infections later in life, thereby conferring a potential advantage to the host.

The above-described protection is reminiscent of an innate memory-like mechanism termed “trained immunity” (21). To test if gut-evolved C. albicans strains are efficient immune trainers, we adopted a well-established mouse model based on intravenous priming of WT C57BL/6 mice followed by systemic challenge with a lethal dose of a fully virulent C. albicans strain (SC5314) 28 days later (22). As expected, all mock-vaccinated animals died within 3 to 4 days postchallenge (dpc) (Fig. 4A). Consistent with previous reports (22), priming with a sublethal dose (1 × 104 CFUs) of WT C. albicans SC5314 delayed host mortality, but eventually all animals succumbed to the challenge (fig. S8). By contrast, 60 to 80% of mice primed with a full dose of a w10 strain survived the secondary challenge (Fig. 4A). Importantly, afilamentous efg1−/− and flo8−/− strains also conferred a statistically significant protection, albeit not as efficiently as the gut-evolved strains. Comparing all strains at the lower dose, the evolved strain R24 protected WT and Rag1−/− mice against systemic candidiasis more efficiently than SC5314 (fig. S8, A and B). This enhanced immunity to a secondary challenge could be due to persistence of the primary avirulent strains in the mouse organs (fig. S9). However, the efg1−/− strain protected its host less efficiently against secondary infections even though it colonized the mouse kidneys at least as efficiently as gut-evolved strains. Taken together, these results suggest that filamentation loss increases the ability of C. albicans to boost host immunity but that gut-evolved strains likely carry additional modifications that further enhance this ability.

Fig. 4 Gut-evolved C. albicans strains confer hosts with broadly cross-protective innate immunity against a wide range of pathogens.

(A) WT mice systemically primed with w10 evolved strains are significantly protected from systemic candidiasis. Survival is significantly higher in these mice than in mice primed with efg1−/− or flo8−/− mutants (shown here) or with a sublethal dose of WT C. albicans cells (fig. S8A). n = 10 mice per group. (B) Rag1−/− mice primed with most w10 evolved strains are significantly protected from systemic candidiasis. In most cases, survival is higher than that of mice primed with the efg1−/− mutant or with a sublethal dose of WT C. albicans cells (fig. S8B). n = 5 to 10 mice per group. In (A) and (B), w10 evolved strains harbored (filled circles) or did not harbor (empty circles) FLO8-inactivating mutations. (C) WT mice primed with a w10 evolved strain (R24) are significantly protected from systemic candidiasis as early as 1 dpp. n = 10 mice per group. (D) Serum IL-6 concentrations of WT mice infected with live w10 strains (W2N or R24) strains are increased at 7 dpp compared with mice infected with a sublethal dose of live (104 CFUs) or a full dose of heat-killed (HK) WT (SC5314). n = 4 to 8 mice per group. (E) Kidney IL-6 amounts, normalized based on organ weight, of WT mice are increased both at 2 and 28 dpp. n = 4 to 17 mice per group. (F) Splenocytes extracted from WT mice 28 dpp with a w10 strain (W2N or R24) produce higher amounts of IL-6 upon ex vivo stimulation with HK-WT SC5314. By contrast, the efg1−/− mutant fails to significantly train splenocytes. Cytokines concentrations were measured 48 hours poststimulation. n = 6 to 10 mice per group. Data are means ± SD. (G) 50% of mice primed with a w10 strain (R24) succumbed after systemic challenge by WT SC5314 when treated with an IL-6 neutralizing antibody but not a control antibody. n = 7 to 13 mice per group. (H to J) WT mice primed with a w10 strains (W2N or R24) are significantly protected from systemic challenge with A. fumigatus (H), S. aureus (I), or P. aeruginosa (J). n = 10 to 11 mice per group. In many cases, survival is significantly higher than that of mice primed with the efg1−/− mutant or a sublethal dose of WT SC5314. Data were analyzed by log-rank test [(A) to (C) and (G) to (J)] or Mann-Whitney test [(D) to (F)]. For (A) to (J), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Because the increased protection observed in w10-primed mice over those immunized with sublethal doses of WT C. albicans correlated with increased total as well as anti–C. albicans–specific immunoglobulin G titers in the serum (fig. S10), we repeated above experiments in Rag1−/− mice to test the contribution of adaptive immunity to this protective mechanism. Similar to WT mice, Rag1−/− mice immunized with w10 strains were significantly more protected from systemic candidiasis than nonimmunized mice. Again, immunization with afilamentous efg1−/− or flo8−/− strains significantly delayed mortality, although not as efficiently as some of the w10 strains (Fig. 4B). Also gut-colonized Rag1−/− pups were significantly protected against systemic candidiasis once they reached adult age, with SC5314-colonized mice achieving ~30% survival and R24-colonized mice >40% survival at 35 dpc (fig. S11). Overall, these data indicate that gut-evolved C. albicans strains raise protective immune responses independently of B and T cells.

Whereas adaptive immunity typically requires a few weeks to mount long-lived memory responses, innate immune responses are characterized by a more rapid onset but shorter lifetimes (23). Consistent with an innate-like mechanism, significant protection against infection with a fully virulent C. albicans strain was achieved as early as 1 day postpriming (dpp) with the R24 gut-evolved C. albicans strain (Fig. 4C) and at 3 months postpriming, whereas most w10 strains still conferred WT mice with a significant protection against systemic candidiasis, mouse survival was significantly reduced compared to mice challenged 1 month postpriming with most w10 strains (fig. S8C). Moreover, Rag1−/− mice show some protection 3 months after immunization, although this effect was no longer significant for some w10 strains (fig. S8D), suggesting that the innate immune effect could last at least until this time point.

Cytokines play crucial roles in innate immune responses (21). Consistently, w10 strains R24 or W2N induced a significant increase in circulating interleukin-6 (IL-6) concentrations 7 dpp, similar to that induced by efg1−/− cells (Fig. 4D). Unlike efg1−/− cells, however, both gut-evolved strains also increased kidney IL-6 concentrations at 28 dpp (Fig. 4E), as well as IL-6 and tumor necrosis factor–α (TNF-α) production capacity of ex vivo restimulated splenocytes (Fig. 4F and fig. S12). Importantly, kidney IL-6 amounts, albeit increased compared to mock-infected animals, were at least 10-fold lower than those measured using a lethal dose of the WT strain (Fig. 4E), and IL-6 serum concentrations returned to physiological levels at 28 dpp (Fig. 4D), thereby limiting the potential collateral damage of chronic systemic inflammation. Moreover, injecting a neutralizing anti–IL-6 antibody significantly reduced R24-induced protection against systemic candidiasis (Fig. 4G), demonstrating a crucial role for this cytokine in this protective immune response.

The non-antigen-specific nature of innate immunity predicts that priming with a w10 strain should confer broad cross-protection against a wide range of pathogens. To test this, we first intravenously primed naïve mice with a w10 strain and then challenged them systemically with a lethal dose of the unrelated fungus A. fumigatus; a Gram-positive bacterium, Staphylococcus aureus; or a Gram-negative bacterium, Pseudomonas aeruginosa. In all cases, animals immunized with a w10 strain were significantly protected from infection compared with both naïve mice and those immunized with a sublethal dose of WT C. albicans (Fig. 4, H to J). Moreover, W2N and R24 protected mice significantly better than efg1−/− cells from systemic A. fumigatus challenge (Fig. 4H), and R24 protected them more efficiently than efg1−/− cells from P. aeruginosa infection (Fig. 4J). Confirming that this cross-protection was independent of adaptive immunity, Rag1−/− mice were also protected from systemic aspergillosis after immunization with R24 (Fig. 4H).

Altogether, gut-evolved w10 strains were able to raise protective immune responses characterized by rapid onset, short lifetime, increased innate cytokine responses, pathogen-aspecificity, and independence from T and B cells; thus, this protection mechanism resembles several critical aspects of trained immunity (21), although other innate immunity mechanisms cannot be discounted. This experimental system hence represents a highly efficient method to generate fungal strains that are not only less pathogenic but also highly immunogenic. These evolved C. albicans strains could therefore lead toward safe and effective universal vaccines with a broad spectrum of cross-protection (24) and with activity also in individuals with impaired adaptive immunity, such as HIV/AIDS patients or transplant recipients.

Taken together, these findings indicate that adaptive evolution of a microbe in the mammalian gut can alter a host-pathogen interaction into a mutually advantageous relationship, in which both the microbe and the host gain some benefit from their interaction. While the microbe enhances its competitive fitness in the host GI tract, the host augments (albeit temporarily) its resistance against a wide variety of pathogens, though long-term consequences on host fitness could not be investigated here. A similar observation was recently reported in laboratory worms (25); however, these organisms lack adaptive immunity and a complex gut microbiome. Our data demonstrate that, although adaptive immunity does not influence this innate selection mechanism, the presence of unperturbed bacterial microbiota interferes with this evolutionary process by shifting the balance between selective forces: Selection by gut bacteria outweighs that by the host, and the filamentation program upturns from a fitness burden to a selective advantage. This model explains why, in its natural niche—the human GI tract, where it is heavily outnumbered by commensal bacteria—C. albicans cannot afford to lose this important competition mechanism and why the “virulence” of this pathobiont often emerges only after antibiotic use (26), when this program is unintentionally redirected from the microbiota to the host.

Supplementary Materials

Materials and Methods

Figs. S1 to S12

Tables S1 to S6

References (2939)

References and Notes

Acknowledgments: We thank A. Tan, Y. S. Lim, and N. Tay for technical assistance; Singapore Immunology Network (SIgN) Mouse Core for providing mice; IMCB Advanced Molecular Pathology Laboratory for histology service; C. B. Ong for veterinary pathology evaluation; J. Berman for C. albicans strains; and M. G. Netea, Y. Wang, and G. De Libero for scientific discussions and feedback on the manuscript. Sequencing was performed by the Genome Institute of Singapore Genome Technology and Biology Group, Singapore. This study was supported by Agency for Science, Technology, and Research (A*STAR) Investigatorship awards JCO/1437a00117 to N.P. and JCO/1437a00119 to G.R. and by core funding from SIgN. Author contributions: G.H.W.T. and N.P. designed the study; K.G.S. and F.Z. prepared sequencing libraries; G.H.W.T., X.S., J.A.R.-C., A.S.M.T., T.G.T., G.T.T.L., and G.C.L. performed all other experiments; G.R. designed and supervised cloning experiments; M.Y., W.L., M.P., G.H.W.T., and N.P. analyzed sequencing data; G.H.W.T., J.A.R.-C., and N.P. analyzed all other data and wrote the manuscript; and N.P. supervised the project. Competing interests: G.H.W.T., X.S., and N.P. are inventors on Singapore patent application no. 10201702472T and International PCT application no. PCT/SG2018/050142 submitted by A*STAR that cover methods of generating attenuated fungi and uses thereof. Data and materials availability: The gut-evolved C. albicans strains are available from N.P. under a material transfer agreement with SIgN, A*STAR. Sequencing data has been deposited at NCBI under SRA accession number SRP116719. All other raw data and code can be accessed at (27).

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