In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores.

The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cells. We demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection. IMPORTANCE: Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.

Cytotoxic rhamnolipid micelles drive acute virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that has developed multi- or even pan-drug resistance toward most frontline and last resort antibiotics, leading to increasing frequency of infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment, P. aeruginosa produces numerous virulence factors that contribute to host tissue damage and immune evasion, promoting bacterial colonization and pathogenesis. In this study, we demonstrate the importance of rhamnolipid production in host-pathogen interactions. Secreted rhamnolipids form micelles that exhibited highly acute toxicity toward murine macrophages, rupturing the plasma membrane and causing organellar membrane damage within minutes of exposure. While rhamnolipid micelles (RMs) were particularly toxic to macrophages, they also caused membrane damage in human lung epithelial cells, red blood cells, Gram-positive bacteria, and even noncellular models like giant plasma membrane vesicles. Most importantly, rhamnolipid production strongly correlated with P. aeruginosa virulence against murine macrophages in various panels of clinical isolates. Altogether, our findings suggest that rhamnolipid micelles are highly cytotoxic virulence factors that drive acute cellular damage and immune evasion during P. aeruginosa infections.

Cytotoxic rhamnolipid micelles drive acute virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that has developed multi- or even pan-drug resistance towards most frontline and last resort antibiotics, leading to increasing infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment, P. aeruginosa produces numerous virulence factors that contribute to host tissue damage and immune evasion, promoting bacterial colonization and pathogenesis. In this study, we demonstrate the importance of rhamnolipid production in host-pathogen interactions. Secreted rhamnolipids form micelles that exhibited highly acute toxicity towards murine macrophages, rupturing the plasma membrane and causing organellar membrane damage within minutes of exposure. While rhamnolipid micelles (RMs) were particularly toxic to macrophages, they also caused membrane damage in human lung epithelial cells, red blood cells, Gram-positive bacteria, and even non-cellular models like giant plasma membrane vesicles. Most importantly, rhamnolipid production strongly correlated to P. aeruginosa virulence against murine macrophages in various panels of clinical isolates. Altogether, our findings suggest that rhamnolipid micelles are highly cytotoxic virulence factors that drive acute cellular damage and immune evasion during P. aeruginosa infections.

Examining Sporadic Cancer Mutations Uncovers a Set of Genes Involved in Mitochondrial Maintenance.

Mitochondria are key organelles for cellular health and metabolism and the activation of programmed cell death processes. Although pathways for regulating and re-establishing mitochondrial homeostasis have been identified over the past twenty years, the consequences of disrupting genes that regulate other cellular processes, such as division and proliferation, on affecting mitochondrial function remain unclear. In this study, we leveraged insights about increased sensitivity to mitochondrial damage in certain cancers, or genes that are frequently mutated in multiple cancer types, to compile a list of candidates for study. RNAi was used to disrupt orthologous genes in the model organism Caenorhabditis elegans, and a series of assays were used to evaluate these genes' importance for mitochondrial health. Iterative screening of ~1000 genes yielded a set of 139 genes predicted to play roles in mitochondrial maintenance or function. Bioinformatic analyses indicated that these genes are statistically interrelated. Functional validation of a sample of genes from this set indicated that disruption of each gene caused at least one phenotype consistent with mitochondrial dysfunction, including increased fragmentation of the mitochondrial network, abnormal steady-state levels of NADH or ROS, or altered oxygen consumption. Interestingly, RNAi-mediated knockdown of these genes often also exacerbated α-synuclein aggregation in a C. elegans model of Parkinson's disease. Additionally, human orthologs of the gene set showed enrichment for roles in human disorders. This gene set provides a foundation for identifying new mechanisms that support mitochondrial and cellular homeostasis.

Pathogen-induced dormancy in liquid limits gastrointestinal colonization of Caenorhabditis elegans.

Colonization is generally considered a prerequisite for infection, but this event is context-dependent, as evidenced by the differing ability of the human pathogen Pseudomonas aeruginosa to efficiently colonize Caenorhabditis elegans on agar but not in liquid . In this study, we examined the impact of the environment, pathogen, host, and their interactions on host colonization. We found that the transition to a liquid environment reduces food uptake by about two-fold. Also expression of specific adhesins was significantly altered in liquid-based assays for P. aeruginosa, suggesting that it may be one factor driving diminished colonization. Unexpectedly, host immune pathways did not appear to play a significant role in decreased colonization in liquid. Although knocking down key immune pathways (e.g. daf-16 or zip-2), either alone or in combination, significantly reduced survival, the changes in colonization were very small. In spite of the limited bacterial accumulation in the liquid setting, pathogenic colonization was still required for the virulence of Enterococcus faecalis. In addition, we found that a pathogen-induced dormancy was displayed by C. elegans in liquid medium after pathogen exposure, resulting in cessation of pharyngeal pumping and a decrease in bacterial intake. We conclude that poor colonization in liquid is likely due to a combination of environmental factors and host-pathogen interactions. These results provide new insights into mechanisms for colonization in different models, enabling pathogenesis models to be fine-tuned to more accurately represent the conditions seen in human infections so that new tools for curbing bacterial and fungal infections can be developed.

In vitro Lung Epithelial Cell Model Reveals Novel Roles for Pseudomonas aeruginosa Siderophores.

Multidrug-resistant Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model to characterize the impact and molecular mechanism of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model for human bronchial epithelial cells (16HBE). We demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipid factors or genetic disruption of rhamnolipid biosynthesis was sufficient to abrogate conditioned medium toxicity. Furthermore, we also examine the effects of purified pyoverdine exposure on 16HBE cells. While pyoverdine accumulated within cells, the siderophore was largely sequestered within early endosomes, showing minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several proinflammatory genes. However, pyoverdine potentiated these iron chelators in activating proinflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection.

The emergent role of mitochondrial surveillance in cellular health.

Mitochondrial dysfunction is one of the primary causatives for many pathologies, including neurodegenerative diseases, cancer, metabolic disorders, and aging. Decline in mitochondrial functions leads to the loss of proteostasis, accumulation of ROS, and mitochondrial DNA damage, which further exacerbates mitochondrial deterioration in a vicious cycle. Surveillance mechanisms, in which mitochondrial functions are closely monitored for any sign of perturbations, exist to anticipate possible havoc within these multifunctional organelles with primitive origin. Various indicators of unhealthy mitochondria, including halted protein import, dissipated membrane potential, and increased loads of oxidative damage, are on the top of the lists for close monitoring. Recent research also indicates a possibility of reductive stress being monitored as part of a mitochondrial surveillance program. Upon detection of mitochondrial stress, multiple mitochondrial stress-responsive pathways are activated to promote the transcription of numerous nuclear genes to ameliorate mitochondrial damage and restore compromised cellular functions. Co-expression occurs through functionalization of transcription factors, allowing their binding to promoter elements to initiate transcription of target genes. This review provides a comprehensive summary of the intricacy of mitochondrial surveillance programs and highlights their roles in our cellular life. Ultimately, a better understanding of these surveillance mechanisms is expected to improve healthspan.

Long-Term Dominance of Carbapenem-Non-Susceptible Pseudomonas aeruginosa ST111 in Hematologic Malignancy Patients and Hematopoietic Cell Transplant Recipients.

An epidemiological study uncovered that fluoroquinolone (FQ) neutropenic prophylaxis in hematopoietic cell transplant and hematologic malignancy (HCT/HM) patients was associated with breakthrough Pseudomonas aeruginosa bloodstream infections (BSIs) with isolates non-susceptible to both FQs and meropenem. The molecular epidemiology of the FQ/meropenem-non-susceptible P. aeruginosa isolates causing FQ-breakthrough BSIs in the HCT/HM patients remains unclear. Through whole genome sequencing on 57 P. aeruginosa isolates from 54 patients diagnosed with HM or receiving an HCT, we found that ST111 strains predominated, accounting for 22 (38.6%) of the isolates. 17 of 33 (51.5%) FQ-breakthrough BSIs were caused by ST111 strains, of which 15 (88.2%) were meropenem non-susceptible. ST111 strains, but not other oprD-deficient, meropenem-non-susceptible clinical strains, were found to have a colonization advantage over P. aeruginosa strain PA14 in C. elegans and to outcompete PA14 in in vitro co-culture assays. Together, we found that breakthrough P. aeruginosa BSIs during FQ prophylaxis in HCT/HM patients are dominated by clonally-related FQ/meropenem non-susceptible strains, predominantly ST111 type, and that the dominance of ST111 strains may be explained by a relative fitness advantage over other clinical strains. Additional work is necessary to better understand the factors driving the dominance and persistence of these ST111 strains.

Novel mitochondria-targeting compounds selectively kill human leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous group of aggressive hematological malignancies commonly associated with treatment resistance, high risk of relapse, and mitochondrial dysregulation. We identified six mitochondria-affecting compounds (PS compounds) that exhibit selective cytotoxicity against AML cells in vitro. Structure-activity relationship studies identified six analogs from two original scaffolds that had over an order of magnitude difference between LD50 in AML and healthy peripheral blood mononuclear cells. Mechanistically, all hit compounds reduced ATP and selectively impaired both basal and ATP-linked oxygen consumption in leukemic cells. Compounds derived from PS127 significantly upregulated production of reactive oxygen species (ROS) in AML cells and triggered ferroptotic, necroptotic, and/or apoptotic cell death in AML cell lines and refractory/relapsed AML primary samples. These compounds exhibited synergy with several anti-leukemia agents in AML, acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML). Pilot in vivo efficacy studies indicate anti-leukemic efficacy in a MOLM14/GFP/LUC xenograft model, including extended survival in mice injected with leukemic cells pre-treated with PS127B or PS127E and in mice treated with PS127E at a dose of 5 mg/kg. These compounds are promising leads for development of future combinatorial therapeutic approaches for mitochondria-driven hematologic malignancies such as AML, ALL, and CML.

Box C/D small nucleolar ribonucleoproteins regulate mitochondrial surveillance and innate immunity.

Monitoring mitochondrial function is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start transcription of protective genes. In Caenorhabditis elegans, several systems are known to play this role, including the UPRmt, MAPKmt, and the ESRE pathways. These pathways are highly conserved and their loss compromises survival following mitochondrial stress. In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immune pathways. We showed that box C/D, but not box H/ACA, snoRNPs are required for the full function of UPRmt and ESRE upon stress. The loss of box C/D snoRNPs reduced mitochondrial mass, mitochondrial membrane potential, and oxygen consumption rate, indicating overall degradation of mitochondrial function. Concomitantly, the loss of C/D snoRNPs increased immune response and reduced host intestinal colonization by infectious bacteria, improving host resistance to pathogenesis. Our data may indicate a model wherein box C/D snoRNP machinery regulates a "switch" of the cell's activity between mitochondrial surveillance and innate immune activation. Understanding this mechanism is likely to be important for understanding multifactorial processes, including responses to infection and aging.

Mitochondria-affecting small molecules ameliorate proteostasis defects associated with neurodegenerative diseases.

Macroautophagic recycling of dysfunctional mitochondria, known as mitophagy, is essential for mitochondrial homeostasis and cell viability. Accumulation of defective mitochondria and impaired mitophagy have been widely implicated in many neurodegenerative diseases, and loss-of-function mutations of PINK1 and Parkin, two key regulators of mitophagy, are amongst the most common causes of heritable parkinsonism. This has led to the hypothesis that pharmacological stimulation of mitophagy may be a feasible approach to combat neurodegeneration. Toward this end, we screened ~ 45,000 small molecules using a high-throughput, whole-organism, phenotypic screen that monitored accumulation of PINK-1 protein, a key event in mitophagic activation, in a Caenorhabditis elegans strain carrying a Ppink-1::PINK-1::GFP reporter. We obtained eight hits that increased mitochondrial fragmentation and autophagosome formation. Several of the compounds also reduced ATP production, oxygen consumption, mitochondrial mass, and/or mitochondrial membrane potential. Importantly, we found that treatment with two compounds, which we named PS83 and PS106 (more commonly known as sertraline) reduced neurodegenerative disease phenotypes, including delaying paralysis in a C. elegans ß-amyloid aggregation model in a PINK-1-dependent manner. This report presents a promising step toward the identification of compounds that will stimulate mitochondrial turnover.

Imaging and Fluorescence Quantification in Caenorhabditis eleganswith Flow Vermimetry and Automated Microscopy.

Gene activation and cellular biomarkers are commonly monitored using fluorescent signals from transgenic reporters or dyes. These quantifiable markers are critical for biological research and serve as an incredibly powerful tool, even more so when combined with high-throughput screening. Caenorhabditis elegans is a particularly useful model in this regard, as it is inexpensive to grow in vast numbers, has a rapid generation time, is optically transparent, and can readily fit within 384-well plates. However, fluorescence quantification in worms is often cumbersome. Quantification is frequently performed using laborious, low-throughput, bias-prone methods that measure fluorescence in a comparatively small number of individual worms. Here we describe two methods, flow vermimetry using a COPAS BioSorter and an automated imaging platform and analysis pipeline using a Cytation5 multimode plate reader and image analysis software, that enable high-throughput, high-content screening in C. elegans. Flow vermimetry provides a better signal-to-noise ratio with fewer processing steps, while the Cytation5 provides a convenient platform to image samples across time. Fluorescence values from the two methods show strong correlation. Either method can be easily extended to include other parameters, such as the measurement of various metabolites, worm viability, and other aspects of cell physiology. This broadens the utility of the system and allows it to be used for a wide range of molecular biological purposes.

Pyoverdine Inhibitors and Gallium Nitrate Synergistically Affect Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that frequently causes ventilator-associated pneumonia in intensive care units and chronic lung infections in cystic fibrosis patients. The rising prevalence of drug-resistant bacteria demands the exploration of new therapeutic avenues for treating P. aeruginosa infections. Perhaps the most thoroughly explored alternative is to use novel treatments to target pathogen virulence factors, like biofilm or toxin production. Gallium(III) nitrate is one such agent. It has been recognized for its ability to inhibit pathogen growth and biofilm formation in P. aeruginosa by disrupting bacterial iron homeostasis. However, irreversible sequestration by pyoverdine substantially limits its effectiveness. In this report, we show that disrupting pyoverdine production (genetically or chemically) potentiates the efficacy of gallium nitrate. Interestingly, we report that the pyoverdine inhibitor 5-fluorocytosine primarily functions as an antivirulent, even when it indirectly affects bacterial growth in the presence of gallium, and that low selective pressure for resistance occurs. We also demonstrate that the antibiotic tetracycline inhibits pyoverdine at concentrations below those required to prevent bacterial growth, and this activity allows it to synergize with gallium to inhibit bacterial growth and rescue Caenorhabditis elegans during P. aeruginosa pathogenesis. IMPORTANCE P. aeruginosa is one of the most common causative agents for ventilator-associated pneumonia and nosocomial bacteremia and is a leading cause of death in patients with cystic fibrosis. Pandrug-resistant strains of P. aeruginosa are increasingly identified in clinical samples and show resistance to virtually all major classes of antibiotics, including aminoglycosides, cephalosporins, and carbapenems. Gallium(III) nitrate has received considerable attention as an antipseudomonal agent that inhibits P. aeruginosa growth and biofilm formation by disrupting bacterial iron homeostasis. This report demonstrates that biosynthetic inhibitors of pyoverdine, such as 5-fluorocytosine and tetracycline, synergize with gallium nitrate to inhibit P. aeruginosa growth and biofilm formation, rescuing C. elegans hosts during pathogenesis.

High-Throughput Approaches for the Identification of Pseudomonas aeruginosa Antivirulents.

Antimicrobial resistance is a serious medical threat, particularly given the decreasing rate of discovery of new treatments. Although attempts to find new treatments continue, it has become clear that merely discovering new antimicrobials, even if they are new classes, will be insufficient. It is essential that new strategies be aggressively pursued. Toward that end, the search for treatments that can mitigate bacterial virulence and tilt the balance of host-pathogen interactions in favor of the host has become increasingly popular. In this review, we will discuss recent progress in this field, with a special focus on synthetic small molecule antivirulents that have been identified from high-throughput screens and on treatments that are effective against the opportunistic human pathogen Pseudomonas aeruginosa.

Innate immunity in C. elegans.

In its natural habitat, C. elegans encounters a wide variety of microbes, including food, commensals and pathogens. To be able to survive long enough to reproduce, C. elegans has developed a complex array of responses to pathogens. These activities are coordinated on scales that range from individual organelles to the entire organism. Often, the response is triggered within cells, by detection of infection-induced damage, mainly in the intestine or epidermis. C. elegans has, however, a capacity for cell non-autonomous regulation of these responses. This frequently involves the nervous system, integrating pathogen recognition, altering host biology and governing avoidance behavior. Although there are significant differences with the immune system of mammals, some mechanisms used to limit pathogenesis show remarkable phylogenetic conservation. The past 20 years have witnessed an explosion of host-pathogen interaction studies using C. elegans as a model. This review will discuss the broad themes that have emerged and highlight areas that remain to be fully explored.

Development and Characterization of High-Throughput Caenorhabditis elegans - Enterococcus faecium Infection Model.

The genus Enterococcus includes two Gram-positive pathogens of particular clinical relevance: E. faecalis and E. faecium. Infections with each of these pathogens are becoming more frequent, particularly in the case of hospital-acquired infections. Like most other bacterial species of clinical importance, antimicrobial resistance (and, specifically, multi-drug resistance) is an increasing threat, with both species considered to be of particular importance by the World Health Organization and the US Centers for Disease Control. The threat of antimicrobial resistance is exacerbated by the staggering difference in the speeds of development for the discovery and development of the antimicrobials versus resistance mechanisms. In the search for alternative strategies, modulation of host-pathogen interactions in general, and virulence inhibition in particular, have drawn substantial attention. Unfortunately, these approaches require a fairly comprehensive understanding of virulence determinants. This requirement is complicated by the fact that enterococcal infection models generally require vertebrates, making them slow, expensive, and ethically problematic, particularly when considering the thousands of animals that would be needed for the early stages of experimentation. To address this problem, we developed the first high-throughput C. elegans-E. faecium infection model involving host death. Importantly, this model recapitulates many key aspects of murine peritonitis models, including utilizing similar virulence determinants. Additionally, host death is independent of peroxide production, unlike other E. faecium-C. elegans virulence models, which allows the assessment of other virulence factors. Using this system, we analyzed a panel of lab strains with deletions of targeted virulence factors. Although removal of certain virulence factors (e.g., Δfms15) was sufficient to affect virulence, multiple deletions were generally required to affect pathogenesis, suggesting that host-pathogen interactions are multifactorial. These data were corroborated by genomic analysis of selected isolates with high and low levels of virulence. We anticipate that this platform will be useful for identifying new treatments for E. faecium infection.

Mitochondrial metabolism as a target for acute myeloid leukemia treatment.

Acute myeloid leukemias (AML) are a group of aggressive hematologic malignancies resulting from acquired genetic mutations in hematopoietic stem cells that affect patients of all ages. Despite decades of research, standard chemotherapy still remains ineffective for some AML subtypes and is often inappropriate for older patients or those with comorbidities. Recently, a number of studies have identified unique mitochondrial alterations that lead to metabolic vulnerabilities in AML cells that may present viable treatment targets. These include mtDNA, dependency on oxidative phosphorylation, mitochondrial metabolism, and pro-survival signaling, as well as reactive oxygen species generation and mitochondrial dynamics. Moreover, some mitochondria-targeting chemotherapeutics and their combinations with other compounds have been FDA-approved for AML treatment. Here, we review recent studies that illuminate the effects of drugs and synergistic drug combinations that target diverse biomolecules and metabolic pathways related to mitochondria and their promise in experimental studies, clinical trials, and existing chemotherapeutic regimens.

Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans.

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans' ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.