Bacterial-fungal interactions and their impact on microbial pathogenesis.

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 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 studies that have mined microbes for novel antimicrobials and antivirulence factors, highlighting the potential of the human microbiota as a rich resource for small molecule discovery.

The putative prenyltransferase Nus1 is required for filamentation in the human fungal pathogen Candida albicans.

Candida albicans is a major fungal pathogen of humans that can cause serious systemic infections in vulnerable immunocompromised populations. One of its virulence attributes is its capacity to transition between yeast and filamentous morphologies, but our understanding of this process remains incomplete. Here, we analyzed data from a functional genomic screen performed with the C. albicans Gene Replacement And Conditional Expression collection to identify genes crucial for morphogenesis in host-relevant conditions. Through manual scoring of microscopy images coupled with analysis of each image using a deep learning-based method termed Candescence, we identified 307 genes important for filamentation in tissue culture medium at 37°C with 5% CO2. One such factor was orf19.5963, which is predicted to encode the prenyltransferase Nus1 based on sequence homology to Saccharomyces cerevisiae. We further showed that Nus1 and its predicted interacting partner Rer2 are important for filamentation in multiple liquid filament-inducing conditions as well as for wrinkly colony formation on solid agar. Finally, we highlight that Nus1 and Rer2 likely govern C. albicans morphogenesis due to their importance in intracellular trafficking, as well as maintaining lipid homeostasis. Overall, this work identifies Nus1 and Rer2 as important regulators of C. albicans filamentation and highlights the power of functional genomic screens in advancing our understanding of gene function in human fungal pathogens.

Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1.

Candida albicans is an opportunistic human fungal pathogen and a member of the mucosal microbiota. To survive in the host and cause disease, C. albicans utilizes several virulence traits, including the ability to respond and adapt to diverse stressors, as well as the morphogenetic switch between yeast and filamentous morphologies. While complex cellular circuitry governs these virulence attributes, the following two kinase-mediated signaling pathways play particularly critical roles in controlling these processes: the Hog1 mitogen-activated protein kinase (MAPK) cascade and the protein kinase A (PKA) pathway. Here, we describe the construction of C. albicans strains harboring substitutions in the ATP-binding pockets of Hog1 and the catalytic subunits of PKA, Tpk1, and Tpk2 to render their activities sensitive to the addition of bulky ATP analogs. Specifically, inhibition by the ATP analog 1NM-PP1 resulted in phenotypes characteristic of the corresponding homozygous deletion mutants for each kinase gene. These strains represent a toolset for the rapid and specific inhibition of PKA and Hog1 kinase activity to further understand their roles in regulating C. albicans morphogenesis and stress responses. IMPORTANCE As an opportunistic pathogen in humans, the fungus Candida albicans relies on virulence traits to cause disease. They include the ability to transition from yeast to filamentous morphologies and the ability to grow in diverse environmental stress conditions, including nutrient limitation, as well as osmotic and heat shock. Previous work identified the following two kinases that play a critical role in regulating these responses: Hog1 and PKA. Here, we generated versions of each kinase that are sensitive to inhibition by a bulky ATP analog, 1NM-PP1. In the presence of the analog, kinase activity is inhibited rapidly and specifically, facilitating the analysis of both kinases in regulating C. albicans morphogenesis and stress responses. Together, these strains represent an important toolset to further our understanding of C. albicans biology and virulence.

Functional genomic analysis of Candida albicans protein kinases reveals modulators of morphogenesis in diverse environments.

Candida albicans is a leading cause of mycotic infection. The ability to transition between yeast and filamentous forms is critical to C. albicans virulence and complex signaling pathways regulate this process. Here, we screened a C. albicans protein kinase mutant library in six environmental conditions to identify regulators of morphogenesis. We identified the uncharacterized gene orf19.3751 as a negative regulator of filamentation and follow-up investigations implicated a role for orf19.3751 in cell cycle regulation. We also uncovered a dual role for the kinases Ire1 and protein kinase A (Tpk1 and Tpk2) in C. albicans morphogenesis, specifically as negative regulators of wrinkly colony formation on solid medium but positive regulators of filamentation in liquid medium. Further analyses suggested Ire1 modulates morphogenesis in both media states in part through the transcription factor Hac1 and in part through independent mechanisms. Overall, this work provides insights into the signaling governing morphogenesis in C. albicans.

DYRK-family kinases regulate Candida albicans morphogenesis and virulence through the Ras1/PKA pathway.

IMPORTANCE: Candida albicans is an opportunistic human fungal pathogen that frequently causes life-threatening infections in immunocompromised individuals. To cause disease, the fungus employs several virulence traits, including its ability to transition between yeast and filamentous states. Previous work identified a role for the kinase Yak1 in regulating C. albicans filamentation. Here, we demonstrate that Yak1 regulates morphogenesis through the canonical cAMP/PKA pathway and that this regulation is environmentally contingent, as host-relevant concentrations of CO2 bypass the requirement of Yak1 for C. albicans morphogenesis. We show a related kinase, Pom1, is important for filamentation in the absence of Yak1 under these host-relevant conditions, as deletion of both genes blocked filamentous growth under all conditions tested. Finally, we demonstrate that Yak1 is required for filamentation in a mouse model of C. albicans dermatitis using genetic and pharmacological approaches. Overall, our results expand our understanding of how Yak1 regulates an important virulence trait in C. albicans.

Respiration supports intraphagosomal filamentation and escape of Candida albicans from macrophages.

IMPORTANCE: Candida albicans is a leading human fungal pathogen that often causes life-threatening infections in immunocompromised individuals. The ability of C. albicans to transition between yeast and filamentous forms is key to its virulence, and this occurs in response to many host-relevant cues, including engulfment by host macrophages. While previous efforts identified C. albicans genes required for filamentation in other conditions, the genes important for this morphological transition upon internalization by macrophages remained largely enigmatic. Here, we employed a functional genomic approach to identify genes that enable C. albicans filamentation within macrophages and uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex. Additionally, we showed that glucose uptake and glycolysis by macrophages support C. albicans filamentation. This work provides insights into the metabolic dueling that occurs during the interaction of C. albicans with macrophages and identifies vulnerabilities in C. albicans that could serve as promising therapeutic targets.

A small molecule produced by Lactobacillus species blocks Candida albicans filamentation by inhibiting a DYRK1-family kinase.

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-ß-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-ß-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1.

High-Throughput Screening Identifies Genes Required for Candida albicans Induction of Macrophage Pyroptosis.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

The effects of CO(2) and chronic cold exposure on fecundity of female Drosophila melanogaster.

Carbon dioxide and chilling are sometimes used to immobilise insects for laboratory research. Both of these methods are known to have short-term effects on behaviour and physiology in Drosophila, but their long-term impacts are unknown. We exposed female D. melanogaster adults to high CO(2) concentrations (4h at 18,000ppm) and chronic cold (72h at 4°C). The carbon dioxide exposure increased chill coma recovery time, but did not result in changes in offspring number, sex ratio, or size. By contrast, the cold exposure resulted in fewer, smaller offspring, and resulted in a male-biased sex ratio compared to controls. There was no significant interaction between CO(2) and cold. We conclude that although caution must be used in choosing an immobilisation method, CO(2) appears to have less long-term impact than cold.