Bacterial-Fungal Interactions and Their Impact on Microbial Pathogenesis

Abstract

Microbial communities of the human microbiota exhibit diverse effects on human health and disease. Microbial homeostasis is important for normal physiological functions and changes to the microbiota are associated with many human diseases including diabetes, cancer, and colitis. In addition, there are also many microorganisms that are either commensal or acquired from environmental reservoirs that can cause diverse pathologies. Importantly, the balance between health and disease is intricately connected to how members of the microbiota interact and affect one another’s growth and pathogenicity. However, the mechanisms that govern these interactions are only beginning to be understood. In this review, we outline bacterial-fungal interactions in the human body, including examining the mechanisms by which bacteria govern fungal growth and virulence, as well as how fungi regulate bacterial pathogenesis. We summarize advances in the understanding of chemical, physical, and protein-based interactions, and their role in exacerbating or impeding human disease. We focus on the three fungal species responsible for the majority of systemic fungal infections in humans: Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. We conclude by summarizing recent work in mining microbes for novel antimicrobials and antivirulence factors, highlighting the potential of the human microbiota as a rich resource for small molecule discovery.

Introduction

Microbial communities are ubiquitous throughout nature, occupying diverse ecosystems and exhibiting a range of interactions within and between species, from symbiosis to competition and predation¹. One ecological niche of particular interest is the human body, as diverse collections of bacteria, fungi, archaea, and viruses living on and within this mammalian host interact to govern diverse aspects of human health^2–5. Specifically, these communities of microbes play pivotal roles in maintaining normal physiological functions, as dysbiosis of the microbiota is associated with many diseases, including inflammatory bowel disease⁶, irritable bowel syndrome⁷, diabetes⁸, obesity⁹, and cancer^10,11, as well as allergies^12,13. Such commensal organisms can be acquired through birth, food consumption, and exposure to diverse environments, and thus can have a profound impact on human development and health throughout an individual’s lifetime^14,15. Importantly, members of the microbiota interact with each other as well as with the host to influence diverse phenotypic traits. Yet, despite the importance of these communications, our understanding of how these players interact remains in its infancy.

In addition to those members of the microbiota that play important roles in maintaining human health, there are also many microorganisms capable of causing diverse pathologies. While traditionally human microbiome studies have focused on bacterial species, advancements in sequencing technology have facilitated the identification and characterization of other members of these microbial communities in the human gut^3,16,17, and other anatomical sites^18–20. Among these commensal organisms are opportunistic fungal pathogens. These eukaryotic pathogens are reported to infect over 1 billion people annually, leading to approximately 1.5 million deaths worldwide^21,22. Most fungal pathogens of humans take advantage of local or systemic suppression of the immune system to cause diverse maladies, including but not limited to: superficial skin, hair, and nail infections; chronic fungal lung infections; and systemic infections with mortality rates as high as 90%²³. The three main opportunistic invaders capable of causing systemic disease in humans include species of Candida, Cryptococcus, and Aspergillus. Humans are exposed to some of these organisms through an environmental reservoir, including Aspergillus fumigatus and Cryptococcus neoformans²⁴. Other fungal infections are caused by constituents of the human microbiota, including Candida albicans, which colonizes the gastrointestinal tract, urogenital tract, and skin²⁴. Not surprisingly, bacterial pathogens capable of residing within a human host are also responsible for significant morbidity and mortality in humans, including but not limited to Pseudomonas aeruginosa in the lungs²⁵, Staphylococcus aureus in the nasopharyngeal cavity²⁶, and Clostridioides difficile in the gut²⁷. While recent work has begun to elucidate the role of bacterial-fungal interactions in opportunistic infections^25,28,29, the mechanisms by which these interactions contribute to pathogenesis remain largely unknown. Several recent publications have also reviewed bacterial-fungal interactions in the context of the human host^3,30–38.

The scope of this review will focus on bacterial-fungal interactions and their role in microbial pathogenesis in the context of human health and disease (Figure 1). In recent years, bacterial-fungal interactions have been reviewed. We provide an overview of current work evaluating bacterial-fungal interactions in the human body, including examining the mechanisms by which bacteria govern fungal growth and virulence, as well as how fungi regulate bacterial pathogenesis. We summarize advances in understanding chemical, physical, and protein-based interactions, and their role in exacerbating or impeding human disease. We conclude by exploring the potential of bacterial-fungal interactions as a rich source for antimicrobial discovery and the identification of anti-virulence strategies to thwart infectious disease.

Figure 1: Bacterial-fungal interactions in the human body.

While this is not a comprehensive list of organisms found in the human microbiota, it highlights the key bacterial-fungal interactions summarized in this review, sorted by anatomical site. Figure made with biorender.com.

Modulation of fungal proliferation and virulence by bacteria

Fungal pathogens of humans employ a range of adaptive mechanisms and virulence factors to facilitate growth and survival during infection of the host (Figure 2A). These include secretion of proteases and toxins, as well as switching between diverse morphological states that assist with adhesion, penetration of host tissue, and evasion of the host immune system^39–42. By affecting the virulence of fungal pathogens, commensal and pathogenic bacteria can modulate the ability of fungi to thrive and cause disease in the host, as well as influence the host-response to the fungus.

Figure 2: Microbial traits modulated by inter-kingdom interactions.

A/ Bacteria influence the ability of C. albicans, C. neoformans, and A. fumigatus to proliferate and modulate antifungal susceptibility, with certain inter-kingdom interactions enhancing these phenotypic traits and others impeding proliferation or compound susceptibility. This is examined in vitro by culturing fungi on solid (shown) or liquid medium in the presence or absence of bacteria or bacterial supernatants. It is also examined using mouse models, where the microbiota has been altered. Finally, the impact of bacteria on antifungal susceptibility can be assessed using a variety of approaches, including disc diffusion assays where the ability of bacterial supernatants to increase the efficacy (zone of inhibition) of antifungals can be assessed. Bacteria also modulate diverse virulence traits including C. albicans hyphal formation, A. fumigatus germination, and C. neoformans capsule formation, melanization, and titan cell induction. Finally, bacteria can modulate biofilm formation in C. albicans and A. fumigatus. Displayed are the developmental stages of C. albicans biofilm formation, including adhesion to a solid surface, induction of hyphal morphogenesis, and maturation and dispersal where an extracellular matrix is produced and yeast cells are released. B/ Fungi can influence the ability of P. aeruginosa and S. aureus to proliferate and can modulate antibacterial susceptibility through increasing the expression of efflux pumps in S. aureus. This has been determined through both in vitro co-culture assays on solid or liquid medium (shown) and through in vivo co-culture models of infection. Fungi can also modulate diverse bacterial virulence traits including inhibition of iron acquisition and quorum sensing in P. aeruginosa, as well as alpha toxin production in S. aureus. Finally, fungi can inhibit mono and polymicrobial biofilm formation in both P. aeruginosa and enhance biofilm formation in S. aureus. Figure made with biorender.com.

Candida albicans

C. albicans is a natural member of the human mucosal microbiota and is a commensal in approximately 50% of healthy adults⁴³. As an opportunistic pathogen, C. albicans is responsible for mucosal infections such as oral and vaginal candidiasis, as well as life-threatening systemic disease in immunocompromised individuals^23,44,45. In its host niche, C. albicans interacts with bacterial commensals, which influence diverse fungal phenotypic traits²⁵. Many bacterial species inhibit C. albicans growth, while others secrete factors that inhibit fungal virulence traits, including filamentation and biofilm formation (Figure 2A)^34,46. In contrast, some bacteria promote fungal growth or enhance C. albicans virulence attributes. Naturally, a thorough understanding of the factors and conditions that govern C. albicans commensalism and pathogenesis is critical to understand how this organism can cause disease.

Proliferation

The ability of C. albicans to proliferate in the host relies on the fungus’ ability to adapt to various environmental perturbations including exposure to elevated temperatures, alternative carbon sources, reactive oxygen species, cell wall stressors, and diverse pH ranges⁴⁷. These responses can all be influenced through interactions with commensal and pathogenic bacteria. In the oral and vaginal microenvironments, C. albicans interacts with Lactobacillus spp., which secrete lactic acid and other weak organic acids that inhibit C. albicans proliferation^48,49. Additionally, Lactobacillus spp. secrete cyclic dipeptides that inhibit the growth of the fungus⁵⁰. This particular inter-kingdom interaction appears to be important in maintaining a healthy physiological state as individuals with reduced colonization by key Lactobacillus spp. are at increased risk for vulvovaginal candidiasis^51,52. Beyond Lactobacillus spp., other commensal bacteria found in the oral mucosa inhibit C. albicans growth in co-culture assays, including Actinomyces israelii and P. aeruginosa, as well as high concentrations of Prevotella nigrescens and Porphyromonas gingivalis⁵³. In the gut, C. albicans interacts with bacteria and other members of the microbiota that also influence its ability to proliferate. Recent work explored interactions between C. albicans and the gut commensal Escherichia coli (strain MG1655) in vitro. E. coli was found to secrete a soluble factor that directly kills the fungus in a magnesium-dependent manner⁵⁴, as depletion of magnesium in a C. albicans-E. coli co-culture rescued growth of the fungus⁵⁴. Notably, the authors indicate that the magnesium levels present in serum of healthy humans are well above the depleted levels used in the study. Therefore, for the inter-kingdom interaction to be physiologically relevant in vivo it would need to occur in microenvironments with depleted magnesium, which may occur at the sites of infection.

Morphogenesis

C. albicans undergoes a transition from yeast to filamentous morphologies in response to a variety of host-relevant cues including exposure to serum, nutrient limitation, neutral pH, and elevated temperature⁵⁵. This transition is important for virulence as C. albicans mutants locked in either morphological state are largely avirulent in mouse models of infection^56,57. The current paradigm is that the filamentous, hyphal morphology is important for tissue invasion and adherence, whereas the yeast morphotype is imperative for dissemination. The filamentous form is also associated with additional virulence factors, including secretion of the cytolytic peptide candidalysin, as well as proteases^41,58. Importantly, previous work demonstrated an inverse relationship between filamentous growth and commensalism, as mutations in key regulators of the yeast-to-filament transition lead to enhanced fitness in the mouse gut⁵⁸. Finally, in addition to filamentous states and the standard ‘white’ round-to-oval yeast morphology, C. albicans can also transition into several elongated yeast-like cell types (opaque, grey, and GUT) that exhibit distinct in vitro properties and interactions with the host⁵⁶. Thus, modulating the diverse morphological states adopted by C. albicans can have a profound consequence on its ability to cause disease.

Commensal and pathogenic bacteria play key roles in regulating the C. albicans yeast-to-hyphal transition, with both inhibitors and enhancers of filamentation identified. One key bacterial-fungal interaction occurs between P. aeruginosa and C. albicans in the human lung (Figure 3A). Previous work found that P. aeruginosa can physically adhere to C. albicans filaments in response to the quorum sensing molecule N-(3-Oxododecanoyl)-L-homoserine lactone, leading to fungal cell death^59,60. Notably, production of this quorum sensing molecule increases resistance of C. albicans to the most widely deployed antifungal, fluconazole, by upregulating efflux pump expression and activating stress response pathways⁶¹. The secretion of additional compounds with antifungal activity by P. aeruginosa is also well characterized, including the production of phenazines, which inhibit C. albicans growth at high concentrations⁶², and inhibit hyphal morphogenesis at sub-inhibitory concentrations⁶³. Phenazine production is at the core of the chemical interaction between C. albicans and P. aeruginosa and elicits a myriad of effects on the host. During growth, C. albicans secretes ethanol as a by-product of fermentation, which enhances the production of phenazine compounds, such as pyocyanin, by P. aeruginosa⁶³. Increased phenazine production further increases fungal ethanol secretion in a positive feedback loop by compromising mitochondrial function⁶⁴. To add further complexity to this interaction, the production of phenazines and ethanol affect the host response to both pathogens, as ethanol reduces the ability of macrophages to clear P. aeruginosa⁶⁵, while phenazines cause direct damage to epithelial tissues⁶⁶. Finally, the host detects phenazines through the aryl hydrocarbon receptor (AhR), upregulating antimicrobial defences and proinflammatory cytokines, which lead to the degradation of virulence factors and eventual clearing of the microorganisms⁶⁷. Interactions between P. aeruginosa and C. albicans are also important upon phagocytosis by host immune cells. Co-infection of macrophages with P. aeruginosa and C. albicans decreases fungal survival, reduces fungal escape from macrophages, and reduces C. albicans filamentation in response to host cells relative to macrophage infection with C. albicans alone⁶⁸. This is dependent on the presence of phenazines, as P. aeruginosa strains defective in compound production are unable to enhance fungal killing by macrophages.

Figure 3: Interactions between bacteria and C. albicans modulate virulence of the fungus.

A/ Interactions between C. albicans and P. aeruginosa on the lung epithelium. P. aeruginosa secretes phenazines such as pyocyanin that inhibit C. albicans growth and biofilm formation, as well as the yeast-to-hyphal transition at concentrations below those inhibiting growth. Through a positive feedback loop, the production of ethanol by C. albicans stimulates phenazine production in P. aeruginosa, and phenazines then further increase ethanol production in the fungus through compromising mitochondrial function. P. aeruginosa quorum sensing (QS) molecules, including N-3-oxo-dodecanoyl-L-homoserine lactone, also inhibit the C. albicans yeast-to-hyphal transition. This QS molecule also upregulates efflux pumps in C. albicans leading to increased resistance to the antifungal, fluconazole. Finally, P. aeruginosa can adhere to C. albicans hyphae and directly kill them through the secretion of phenazines. B/ Interactions between C. albicans and Lactobacillus spp. on the vaginal epithelium. Lactobacillus spp. secrete small molecules including hydrogen peroxide, short-chain fatty acids (SCFAs), and weak organic acids (WOAs) such as lactic acid that affect fungal growth. Lactobacillus spp. also secrete 1-acetyl-beta-carboline that inhibits the yeast-to-filament transition, as well as chitinase, which breaks down the fungal cell wall. Finally, Lactobacillus spp. directly compete with C. albicans for adhesion sites on the vaginal epithelium. Figure made with biorender.com.

Over time, C. albicans and P. aeruginosa have evolved to adapt to the negative pressures each pathogen exerts on one another. For example, clinical isolates of C. albicans from the lungs of cystic fibrosis patients have been identified that are resistant to the filament-repressive effects of P. aeruginosa⁶⁹. Genome sequencing of the C. albicans clinical isolates revealed that most of the constitutively filamentous strains harbored mutations in the transcriptional repressor gene NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different cystic fibrosis patients⁶⁹, providing a poignant example of parallel evolution in the context of the human host.

In the oral cavity, C. albicans interacts with the oral commensal and opportunistic pathogen, Streptococcus mutans. This microbe along with other Streptococcus spp. secrete trans-2-decenoic acid, a small molecule that inhibits C. albicans hyphal morphogenesis without affecting fungal growth⁷⁰. S. mutans also secretes mutanobactin A, another small molecule that blocks C. albicans hyphal morphogenesis⁷¹.

Another mucosal site frequently colonized by C. albicans is the human vagina^18,72. Within this environment, C. albicans is the leading cause of vaginal candidiasis⁷³ and interacts with commensal bacteria on the vaginal mucosa, including Lactobacillus spp. (Figure 3B).^74,75. The ability of Lactobacillus spp. to affect C. albicans morphogenesis is dependent on white-opaque cell type switching. The white cell morphology has been best studied for its interaction with Lactobacillus spp. Cellular proteins secreted by this genus include major secreted protein 1 (Msp1), which is readily produced by Lactobacillus rhamnosus GG and acts as a chitinase to break down the fungal cell wall, blocking filamentous growth in white cells⁷⁶. In addition to chitinase, other factors resistant to protease and heat treatment within Lactobacillus-conditioned medium inhibit C. albicans hyphal morphogenesis in white cells^77–79, although the molecular entity responsible for this effect remained elusive for some time. Fortunately, recent work identified a component of the Lactobacillus secretome, 1-acetyl-beta-carboline, that inhibits C. albicans hyphal morphogenesis and biofilm formation via inhibition of the DYRK1-family kinase, Yak1⁸⁰. However, the same conditions that block filamentation in white cells do not block morphogenesis in opaque cells, highlighting the broad phenotypic plasticity C. albicans displays in response to both its environment and inter-kingdom interactions⁸¹.

Streptococcus agalactiae is another bacterium found in the vaginal microenvironment that is commonly isolated from individuals with recurrent vulvovaginal candidiasis⁸². Similar to Lactobacillus, S. agalactiae-conditioned medium inhibits C. albicans hyphal morphogenesis. However, co-inoculation of mice with C. albicans and S. agalactiae increased fungal burden in a mouse model of recurrent vulvovaginal candidiasis due to a decreased Th17 immune response ⁸², highlighting the complex connections between the fungus, bacterium, and host in governing pathogenesis.

C. albicans also resides in the human gut⁸³ where it interacts with commensal and pathogenic bacteria, including E. coli, Salmonella enterica, and Enterococcus faecalis. Previous work found that secretory products found during culture of E. coli biofilms were able to inhibit C. albicans hyphal morphogenesis and the expression of key hyphal-associated genes⁸⁴. Additionally, S. enterica serovar Typhimurium was able to inhibit C. albicans filamentation in a Caenorhabditis elegans model of infection and directly kill C. albicans hyphae through a mechanism that relied on the inositol phosphatase, sopB⁸⁵. Interestingly, C. albicans-conditioned medium increased the expression of sopB and other genes important for S. enterica-mediated hyphal-killing⁸⁵, highlighting a compensatory mechanism adopted by the bacterium to regulate fungal virulence. Finally, the interaction between E. faecalis and C. albicans has been well studied, where early work found that co-infection with both organisms resulted in attenuated virulence in a C. elegans model⁸⁶. E. faecalis secretes EntV, a bacteriocin with antifungal and anti-filamentation activity, which protects C. elegans from infection with C. albicans⁸⁷. Recent work found that post-translational modifications are important for the regulation of EntV, including identifying the requirement for gelatinase activity to cleave EntV into its active form⁸⁸. Many other bacteria have also been reported to produce soluble factors that govern C. albicans morphogenesis, including the opportunistic pathogens C. difficile and Burkholderia cenocepacia, which secrete para-Cresol⁸⁹, and cis-2-dodecnoic acid (BDSF)⁹⁰, respectively.

More broadly in the gut, short-chain fatty acids (SCFAs) are metabolites produced in the colon by bacterial fermentation of dietary fibers, and these lipids play a key role in maintaining a healthy microbiota. SCFAs inhibit growth, filamentation, and biofilm formation of C. albicans in vitro⁹¹. Consequently, antibacterial-treated mice susceptible to C. albicans gut infection exhibit significantly reduced levels of SCFAs in the cecum and higher fungal loads in the feces⁹¹, providing in vivo evidence that SCFAs are important in controlling C. albicans overgrowth. Additionally, recent work assessed the role of C. albicans in governing pathogenicity in a chemical-induced colitis mouse model, and observed that vaccination with the Candida NDV-3A vaccine protected mice from fungal-induced damage during colitis⁹². In the future, it will be interesting to see if these protective effects are also observed in a bacterial-induced colitis model.

In contrast to the examples highlighted above, commensal bacteria are also implicated in enabling C. albicans morphogenesis in the mammalian gut⁹³. Serial passage of C. albicans through the gastrointestinal tracts of antibiotic-treated mice led to the rapid generation of low-virulence strains unable to form hyphae due to mutations in a gene encoding a transcription factor that positively regulates filamentation, FLO8⁹³. These evolved lineages stimulated proinflammatory cytokines and conferred transient cross-protection against several other gut inhabitants. However, if an intact microbiota was present, only the virulent hyphal form persisted, suggesting that bacterial commensals play a critical role in promoting the filamentous or virulent form. The reason for this contradiction remains unclear but may be due to the commensal status of organisms as opposed to inter-kingdom interactions in disease contexts.

Biofilm Formation

Another important C. albicans virulence trait is its ability to form intrinsically drug-resistant biofilms, or surface-associated communities, which colonize medical devices such as catheters in healthcare settings⁹⁴. While the yeast-to-filament transition is intricately linked to the ability of C. albicans to form these structures, there are numerous other facets that contribute to their formation. C. albicans is frequently isolated in polymicrobial biofilms that are also comprised of Streptococcus spp., P. aeruginosa, and S. aureus^95–97. These primarily occur on healthcare devices such as catheters and pacemakers, as well as in the oral cavity, where co-colonization with bacteria is associated with dental cavities, periodontitis, and denture stomatitis⁹⁸. In the mouth, C. albicans adheres to oral Streptococcus spp., providing additional surface for fungal colonization⁹⁹. Streptococcus gordonii, a common commensal of the oral mucosa, exerts physical force and produces chemical signals that lead to enhanced fungal morphogenesis and biofilm formation in C. albicans¹⁰⁰. Additional work found that the competence regulation system ComDE in S. gordonii is important in the early stages of dual-species biofilms, but inhibits C. albicans biofilm formation in later stages¹⁰¹. Streptococcus oralis colonization of the oral mucosa also leads to increased biofilm formation, enhanced dissemination of C. albicans in mice, and increased expression of proinflammatory cytokines that result in enhanced tissue inflammation and immunopathogenesis¹⁰². Other bacterial members of the oral microbiota also enhance C. albicans virulence in early stages of biofilm formation, including Streptococcus sanguinis, Actinomyces odontolyticus, and Actinomyces viscosus¹⁰³.

In contrast, the opportunistic pathogen and oral commensal Aggregatibacter actinomycetemcomitans secretes the quorum sensing molecule autoinducer-2 that inhibits C. albicans biofilm formation by blocking filamentation¹⁰⁴. As well, P. aeruginosa secretes factors that inhibit C. albicans biofilm formation, although through a mechanism that is independent of morphogenesis¹⁰⁵. Using a strain of P. aeruginosa that does not secrete homoserine lactone, it was discovered that the bacterial supernatant could inhibit biofilm formation in the constitutively filamentous tup1 deletion strain of C. albicans, implying an effect beyond inhibition of hyphal morphogenesis¹⁰⁵.

Cryptococcus neoformans

While studies examining fungal–bacterial interaction have mainly been performed with C. albicans, the impact of bacteria on C. neoformans growth and virulence has also been investigated. C. neoformans is an opportunistic human fungal pathogen and causative agent of cryptococcosis¹⁰⁶. While immunocompromised individuals are most vulnerable to cryptococcal infections, there are also reports of C. neoformans causing systemic infections in immunocompetent hosts^107,108. An estimated 223,100 cases of cryptococcal meningitis occur globally each year, leading to 181,100 deaths¹⁰⁹. These staggeringly high mortality rates are due to numerous factors including a limited antifungal arsenal, the frequent development of antifungal resistance, and the fact that these infections predominantly occur in resource-poor settings where proper medical care is inadequate. As a human fungal pathogen, C. neoformans relies on several unique virulence traits to survive in a human host including the ability to proliferate at mammalian body temperature, as well as the capacity to form a polysaccharide capsule, produce melanin, and form the atypical titan cell morphology (Figure 2)¹¹⁰. Titan cells are cryptococcal cells with enormous dimensions and clinical relevance due in part to being refractory to phagocytosis by human immune cells¹¹¹.

Proliferation

Given the diverse environmental niches that C. neoformans is capable of inhabiting, there are many reports of diverse bacterial species exerting anti-cryptococcal activity³¹. C. neoformans is commonly isolated from pigeon guano suggesting that its gastrointestinal tract is at least temporarily colonized with this fungus, despite this species being recalcitrant to cryptococcal infection¹¹². Early work that co-incubated C. neoformans with seven species of bacteria found within the pigeon microbiota observed a complete inhibition of C. neoformans growth, speculating that a specialized avian microbiota may at least partially protect birds from infections by C. neoformans¹¹³. Follow-up investigations found that the growth-inhibitory activity was mainly exerted by only two bacterial species, P. aeruginosa and Bacillus subtilis¹¹⁴. P. aeruginosa can inhibit C. neoformans growth through both a contact-dependent mechanism as well as contact-independent mechanisms that include the secretion of pyocyanin and other phenazine derivatives¹¹⁵. S. aureus is also reported to kill C. neoformans through a mechanism that involves attachment to the capsule¹¹⁶ as the anti-proliferative effects are specific to C. neoformans and not to other fungal pathogens that do not produce the polysaccharide layer. While these examples all highlight inter-kingdom interactions that impair C. neoformans growth, the soil bacterium Acinetobacter baumanii was observed to increase C. neoformans survival in biofilms and stimulate the formation of capsule¹¹⁷. The exact molecular mechanism of this interaction remains to be determined; however, physical contact was not required, at least for the biofilm-inducing activity, suggesting that A. baumanii likely secretes specific factors that affect the fungus either at the cell surface or inside the cell. Overall, the impact of these inter-kingdom interactions on C. neoformans proliferation is highly complex and dependent on the organism involved.

Virulence

C. neoformans melanization is an important virulence trait to help protect the fungus from oxidative damage, antifungal assault, and high temperature, while also functioning to modulate host immune responses¹¹⁸. To better understand how bacteria regulate C. neoformans melanin production, a screen was performed using 40 microorganisms found in environmental niches occupied by the fungus¹¹⁹. This work identified several species of the Bacillus genus that were able to inhibit melanization without affecting growth. Bacillus safensis was further investigated and found to inhibit other virulence traits including capsule production and biofilm formation, in part via the action of chitinase activity¹¹⁹. There are also examples of bacteria enhancing C. neoformans virulence. The opportunistic bacterial pathogen Klebsiella aerogenes promotes melanization of C. neoformans cells during co-cultivation through the bacterial production of dopamine, a precursor for cryptococcal melanin biosynthesis¹²⁰. Finally, it was recently shown that the mouse microbiota has the capacity to induce titan cell formation by C. neoformans¹²¹. The in vivo significance of the microbiota in promoting titan cell formation was established by observing that mice pre-treated with antibiotics prior to infection with C. neoformans had significantly less fungal cells with the titan morphology compared to antibiotic-free mice¹²¹. Further analysis of the titan cell-inducing mechanisms revealed that bacteria such as E. coli, and Streptococcus pneumoniae trigger cryptococcal titanization via shedding of peptidoglycan, a component of the bacterial cell wall¹²¹.

Aspergillus fumigatus

Aspergillus fumigatus is a saprotrophic fungus ubiquitous in the environment and a leading cause of invasive aspergillosis¹²². A. fumigatus is a significant cause of invasive infections in individuals with impaired immune function, including those with neutropenia, solid organ transplant recipients, and patients on immunosuppressive therapies, such as high-dose corticosteroids. It is estimated that more than 200,000 cases of invasive aspergillosis occur each year, with staggering mortality rates of up to 50% with treatment and 100% if left undiagnosed²⁴. A. fumigatus relies on several virulence traits during infection of a human host, including the production of gliotoxin and germination of conidia into hyphae¹²³.

One of the most consequential bacterial-fungal interactions occurs between A. fumigatus and P. aeruginosa, which can be deadly in individuals with chronic lung conditions, such as cystic fibrosis^97,124. It is well established that P. aeruginosa secretes antifungal compounds with activity against A. fumigatus, including pyocyanin and other phenazines^125,126. Expanding beyond these well-characterized secreted compounds, Sass et al. evaluated 24 P. aeruginosa mutants with deletions in genes important for virulence¹²⁷. The authors found that the P. aeruginosa siderophore pyoverdine was effective at inhibiting A. fumigatus biofilm formation. By capturing extracellular iron, the authors predicted that pyoverdine limits A. fumigatus growth and biofilm formation by creating a nutrient-limited environment¹²⁷. Pyoverdine was later found to work synergistically with the Pseudomonas quinolone signal (PQS) quorum sensing molecule that is also responsible for iron chelation and inhibition of biofilm formation under low iron conditions¹²⁸. Paradoxically, under high iron conditions, PQS enhances A. fumigatus biofilm formation and this process is dependent on the A. fumigatus iron siderophore, ferricrocin¹²⁸. Finally, P. aeruginosa produces volatiles that stimulate A. fumigatus to invade the lung parenchyma when the two organisms are physically separated¹²⁹. However, as soon as the organisms come into direct contact, their relationship becomes antagonistic as they compete for nutrients, including iron¹²⁹. This highlights the complex inter-kingdom interactions displayed between these two opportunistic pathogens that have a profound impact on the human host, as well as the key role of iron in modulating this dynamic relationship.

In other mucosal sites of the human body, an important fungal-bacterial interaction occurs between A. fumigatus and S. aureus. During polymicrobial biofilm formation, S. aureus inhibits A. fumigatus conidiation, filamentation, and biofilm maturation¹³⁰. Another study found that E. coli DH5α secretes a 60 kDa protein with activity against A fumigatus. This activity was then linked to a siderophore-based inhibition of fungal growth via limitation of iron acquisition¹³¹.

Overall, these studies demonstrate that different bacteria have disparate effects on C. albicans, C. neoformans, and A. fumigatus either promoting or preventing growth, and either enhancing or blocking the production of virulence factors. As further studies continue to explore the inter-kingdom interactions that occur in the human host, more mechanistic insights will be gleaned as to how fungal pathogenesis is impacted by other inhabitants of the human microbiota.

Modulation of bacterial proliferation and virulence by fungi

Similar to fungi, bacteria employ a range of virulence traits to facilitate infection in the host, including the use of secretion systems for the release of toxins and other effectors^132,133. Some fungi exhibit broad effects on the virulence of multiple bacterial pathogens. For example, C. albicans biofilms create a hypoxic microenvironment that facilitates the growth of anaerobic bacteria including Clostridium perfringens and Bacteroides fragilis¹³⁴. Here, we summarize current research efforts to evaluate the effect of fungi on bacterial growth and virulence in the context of the human host.

Pseudomonas aeruginosa

P. aeruginosa is a leading cause of hospital-acquired infections, including pneumonia and urinary and wound infections¹³⁵, and is frequently detected in the lungs of cystic fibrosis patients⁹⁷. In fact, over 75 % of CF patients over 18 years of age are chronically colonized with P. aeruginosa, which often persists throughout the life of the patient. Previous work found that C. albicans impacts P. aeruginosa virulence, biofilm formation, and secretion of antifungal compounds. Specifically, C. albicans produces the quorum sensing molecule farnesol, which inhibits transcription from the pqsA–E operon, blocks production of the quinolone signal PQS, and ultimately inhibits the expression of phenazine biosynthetic genes¹³⁶. However, in P. aeruginosa–C. albicans biofilms, where P. aeruginosa and PQS concentrations are high, the presence of the fungus leads to increased production of phenazines through an uncharacterized pathway, suggesting that the effects of C. albicans on P. aeruginosa are complex^62,137. Interestingly, C. albicans can also impact P. aeruginosa virulence by inhibiting iron acquisition. C. albicans-secreted proteins inhibit the expression of P. aeruginosa genes important for iron acquisition and virulence, including pyochelin and pyoverdine¹³⁸. Oral administration of C. albicans secreted proteins was sufficient to protect mice from P. aeruginosa infection and oral iron supplementation rescued bacterial virulence in the presence of C. albicans. P. aeruginosa and A. fumigatus are also frequent co-colonizers in the lungs of cystic fibrosis patients⁹⁷ and gliotoxin produced by A. fumigatus inhibits P. aeruginosa growth and biofilm formation¹³⁹. Other work found that A. fumigatus secretes isocyanides that bind copper and exhibit broad-spectrum antimicrobial activity, including activity against P. aeruginosa¹⁴⁰.

Staphylococcus aureus

S. aureus and C. albicans are frequently co-isolated in biofilm-associated diseases such as keratitis and urinary tract and wound infections¹⁴¹. S. aureus adheres to C. albicans hyphae through the adhesins FnpB, SasF, and Atl to facilitate tissue penetration and seed dissemination of the bacteria¹⁴². In a mouse model of oral candidiasis, co-infection of C. albicans and S. aureus results in the establishment of systemic infection as opposed to symptoms of oral candidiasis with the fungus alone or no symptoms with the bacteria alone¹⁴². A mouse model of intra-abdominal infection also found that coinfection with C. albicans and S. aureus results in synergistic lethality^143,144. Interestingly, this synergism is not dependent on the ability of C. albicans to undergo hyphal morphogenesis and non-albicans species of Candida also enhance infection^145,146. Additionally, recent work established that during polymicrobial growth of C. albicans and S. aureus, the fungus elevates extracellular pH to enhance the production of alpha toxin, the major cytotoxic agent released by the bacterium¹⁴⁷. As well, when exposed to the C. albicans quorum sensing molecule farnesol, S. aureus exhibits enhanced tolerance to antimicrobial agents due to increased expression of drug efflux pumps¹⁴⁸.

Other Examples

S. mutans and C. albicans are frequently co-isolated in plaques and biofilms in the oral mucosa, contributing to caries and other tooth decay and damage, particularly in children. While high levels of farnesol can inhibit S. mutans growth, lower concentrations that are found in S. mutans-C. albicans conditioned medium actually increase growth and enhance biofilm formation in S. mutans¹⁴⁹. Farnesol also increases the expression of glucosyltransferases in S. mutans that contribute to the robust exopolysaccharides found in the extracellular matrix of biofilms¹⁴⁹. Thus, there is a dynamic relationship between farnesol production and S. mutans growth and virulence.

To investigate the mechanisms underlying the high rate of mucosal and systemic candidiasis in cancer patients receiving chemotherapy¹⁵⁰, Bertolini et al. developed a chemotherapy-immunosuppressed mouse model of oral and gut mucosal breach by C. albicans¹⁵¹. The authors showed that infection with C. albicans led to changes in the oral mucosa that contributed to disease¹⁵¹. These changes in the microbial community led to an increase in prevalence of Enterococcus spp., which reduced the integrity of the epithelial barrier and promoted invasion of C. albicans and immunopathology associated with candidiasis.

In contrast to most of the mechanistic studies published over the past decade, recent work established that positive interactions between microbes were much more common than previously predicted. This was determined by using a high-throughput co-culture platform that examined over 180,000 different interactions between 20 soil-dwelling bacteria in 40 different environment conditions¹⁵². It would be interesting to use a similar platform in the future to examine bacterial-fungal interactions in a high-throughput manner to systemically evaluate both positive and negative effects on proliferation and virulence.

Targeting Virulence as an Antimicrobial Strategy

Microbe-derived biomolecules, including natural products, are a rich source of antimicrobial compounds^153,154. About 70% of antibacterial agents used in the clinic are of natural product origin, and 97% of these compounds originate from either fungi or bacteria^155,156. However, a vast majority of these antimicrobials target essential gene products or processes required for pathogen viability. A relatively underexplored area of study is the therapeutic potential of anti-virulence compounds to thwart infectious disease^157,158. A challenge of canonical antimicrobial agents is their relatively non-selective inhibition of microbial growth. This leads to negative effects on commensal microbes in the human body and contributes to antimicrobial resistance^159–161. One of the exciting possibilities of anti-virulence strategies to combat disease is the ability to specifically prevent organisms from causing infections, as opposed to directly killing microbes. This reduces selective pressure on the organism to evolve resistance as cells are still able to grow and survive, without employing virulence factors that damage the host¹⁶². Inhibiting virulence factors also extends potential drug targets beyond those involved in essential processes in pathogens. This is especially important in fungi, as these eukaryotic pathogens share many essential processes with their human hosts. However, there are limitations to anti-virulence strategies, as elimination of the pathogen is necessary in the case of many infections. Anti-virulence strategies alone could be employed for common opportunistic infections such as oral and vaginal candidiasis. However, for bloodstream infections or complicated disease, combination therapy could be employed that uses both an antimicrobial and anti-virulence-based strategy¹⁶³.

While this review has described several secreted compounds produced by members of the human microbiota that modulate key virulence attributes, thus far, the only FDA-approved anti-virulence therapeutic to date has used antibodies to bind and neutralize toxins in bacterial pathogens. The first study to demonstrate efficacy of an anti-virulence strategy against a pathogen involved treatment of infant botulism with antibodies purified from adult donors that neutralize the botulism toxin¹⁶⁴. Building on the success of this approach, a separate study successfully employed an anti-virulence treatment in stage three clinical trials to combat recurrent C. difficile infection¹⁶⁵. The authors administered two monoclonal antibodies, actoxumab and bezlotoxumab, that bind and neutralize the C. difficile toxins A and B, respectively. Treatment with both antibodies was associated with a significant reduction in recurrent C. difficile infection in at-risk patients¹⁶⁵. Additionally, another monoclonal antibody, raxibacumab, binds and neutralizes a component of the anthrax toxin and confers improved survival and clinical outcomes following anthrax exposure in rabbits and monkeys¹⁶⁶. Probiotic bacteria have been extensively investigated for their ability to treat infections that lead to microbial dysbiosis, including diarrheal disease and vaginal candidiasis^167,168. However, only one fungal species is established as a treatment for bacterial infection, including diarrhea caused by C. difficile in adults and children. Previous work found that Saccharomyces boulardii CNCM I-745 secretes a 54-kDa protease which digests the C. difficile toxin A¹⁶⁹. Oral administration of the probiotic fungus has no effect on the microbiota of healthy humans, but it can rescue eubiosis of the intestinal microbiota following diarrheal disease¹⁷⁰ and infection with Helicobacter pylori¹⁷¹. These examples highlight the potential of targeting virulence factors to combat C. difficile infections, including the potential application of microbiota-derived factors.

A relatively unexplored and exciting area of current focus is the potential of small molecules to target virulence traits of human fungal pathogens. Several small molecule inhibitors of C. albicans hyphal morphogenesis have been described (Figure 4)^{60,77,172–174}. This includes small molecules secreted by bacteria, such as 1-acetyl-beta-carboline secreted by Lactobacillus spp.⁸⁰ and the C. albicans quorum sensing molecule, farnesol¹⁷⁵. Other work has screened collections of small molecules for activity against C. albicans hyphal morphogenesis. Specifically, a collection of 30,000 small molecules was assessed for their ability to inhibit adhesion of C. albicans to polystyrene plates, identifying a single molecule, filastatin, that inhibits C. albicans adhesion, hyphal morphogenesis, biofilm formation, and fungal virulence in a nematode model of infection¹⁷². Analogous screens also identified biaryl amide compounds that inhibit C. albicans hyphal morphogenesis and prevent virulence in oral and invasive murine models of candidiasis¹⁷⁶, as well as diazaspiro-decane analogs as inhibitors of C. albicans biofilm formation, hyphal morphogenesis, and virulence in both an oral and invasive model of mouse candidiasis¹⁷³. Additionally, a screen of 678 compounds pre-selected based on bioactivity against Saccharomyces cerevisiae identified Tri-Chloro-Salicyanilide (TCSA) as a top compound that blocks C. albicans hyphal morphogenesis and biofilm formation. Through transcriptional profiling, this activity was linked to fungal mitochondrial protein import¹⁷⁷. However, none of these compounds have advanced to a clinical trial. The only anti-virulence strategy to combat fungal infection that is under clinical development is the NDV-3A C. albicans vaccine¹⁷⁸. It was developed using the Als3 adhesin protein that is critical for C. albicans adherence, invasion, and virulence in the host¹⁷⁹. The vaccine has demonstrated efficacy against both systemic and oral candidiasis in murine models^180–182, as well as recurrent vulvovaginal candidiasis in women in a double-blind, placebo controlled clinical trial¹⁷⁸. Thus, although targeting virulence traits represents a promising therapeutic strategy to mitigate infectious diseases, future study is necessary to develop microbiome-derived compounds with efficacy against fungal pathogens.

Figure 4: Bacteria secrete diverse compounds that inhibit the C. albicans yeast-to-hyphal transition.

The small molecules secreted by bacterial species highlight a diverse collection of chemical scaffolds capable of inhibiting hyphal morphogenesis in C. albicans. Figure made with biorender.com.

Bacteria and fungi alike have been mined for the secretion of antimicrobial compounds, including many front-line anti-infective agents used clinically^183,184. However, the current mining of antimicrobials is neither systematic nor comprehensive, and the re-discovery of antimicrobial agents is a significant hurdle that is yet to be overcome¹⁸⁵. Current work is trying to establish efficient pipelines coupled with modern ‘omics’ technology to facilitate natural product discovery in diverse microbial backgrounds. Such advancements could be applied to those organisms living in the environment as well as those found within a human host. Bioinformatics now facilitates the discovery of silenced or cryptic biosynthetic clusters responsible for natural product production in both fungi and bacteria^186–188. This includes recent work that developed a pipeline for identifying natural products from anaerobic fungi using genomics, transcriptomics, proteomics, and metabolomics, highlighting the untapped potential of anaerobic gut fungi as producers of natural products¹⁸⁹. Whole-genome sequencing to identify unexplored biosynthetic gene clusters has also led to the identification of a known antibacterial complestatin and a new antibacterial corbomycin, that bind to bacterial peptidoglycan and inhibit autolysin activity to prevent essential bacterial cell wall remodelling¹⁹⁰. Both compounds were effective in a murine model of skin infection and decreased methicillin-resistant S. aureus burden¹⁹⁰. Finally, leveraging the microbiomes of marine animals and cutting-edge metabolomics and genomic tools, a novel antifungal turbinmicin was discovered that displays potent in vitro and in vivo activity against multidrug-resistant fungal pathogens through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway¹⁹¹. Continued advances in antimicrobial discovery will help scientists and clinicians realize the potential of anti-virulence strategies to thwart infectious disease.

Conclusion

Dissecting the mechanisms underlying inter-kingdom interactions is important for furthering our understanding of human health and disease. The literature outlined in this review demonstrates that microbial interactions, including both antagonistic and synergistic interactions, have important implications for opportunistic infections and beyond. We highlight the extensive literature that describes the effects of bacteria on fungal growth and virulence in the mammalian host, as well as the effect of fungi on bacterial proliferation and virulence. By focusing on opportunistic fungal and bacterial pathogens that colonize the human body, we highlight key microbial interactions that contribute to the onset and severity of diverse infections. Studies highlighted in this review that investigate the synergistic interactions between bacteria and fungi identify their important implications for polymicrobial infections and disease susceptibility. Additionally, work describing antagonistic interactions between bacteria and fungi demonstrate their potential for the identification of novel antimicrobial compounds and strategies to thwart infectious disease. These novel antimicrobial compounds offer a promising reservoir of untapped chemical diversity that remains largely unexplored. Beyond the medical applications of inter-kingdom interactions, characterizing these communications is important for our understanding of the development and maintenance of the human microbiota. Similar to studying other ecological environments, examining the microbial composition of healthy and disease states can further our understanding of these complex communities.

With the rising threat of antimicrobial resistance, the need for novel antibacterial and antifungal agents is reaching a critical point. Improved strategies for mining bacteria and fungi for natural products now enable researchers to revisit ecological bacterial-fungal interactions as a source of novel antimicrobials. Given that many microbes are opportunistic pathogens that rely on virulence traits to cause disease in a human host, small molecules that inhibit microbial virulence represent an exciting area of antimicrobial discovery. With antibody and vaccine-based anti-virulence strategies beginning to gain approval for clinical use, further research is required to expand the available repertoire of microbial virulence inhibitors.