Felipe Hiram Santiago-Tirado

All pathogens rely on dynamic gene expression for host adaptation and disease causation. This is especially important for pathogens that normally are found in the environment, like Cryptococcus neoformans, given that environmental and host conditions are dramatically different. Epigenetic modifications, like those carried out by SET-domain histone lysine methyltransferases (HKMTs), are essential for this genome expression flexibility. While transcription factors and signaling cascades have been well-studied in C. neoformans ’s host adaptation, the role of HKMTs in this process has not been explored. Here, we characterize the gene SET202 , previously reported as a putative HKMT important for growth in host lungs. We show that set202Δ mutants have 50% less phagosomal permeabilization than the wild-type fungi, have phagosomal maturation defects, and poor intracellular survival. Additionally, they are defective in traits promoting virulence, including significantly smaller capsules, less melanin, and poor growth under host-like conditions. These phenotypes contributed to reduced virulence in a murine model of infection but, notably, this defect was host-temperature-dependent. In order to explain mechanistically how SET202 deletion could affect such a wide range of processes, we assessed and demonstrated, for the first time, that Set202 is a true histone-3-lysine 36 methyltransferase, supporting its role in epigenetically modifying the fungal genome to enable adaptation to the hostile host conditions. Consequently, our findings support the study of the biological function of Set202, as it can serve as a potential future therapeutic target for the treatment of infections caused by C. neoformans, and possibly other pathogenic fungi. IMPORTANCE: Epigenetic modifications are essential for a wide variety of processes, from cancer to infectious diseases. In the fungal world, they have been associated with fungal stress response, antifungal resistance, and disease establishment. However, in the environmental opportunistic pathogen Cryptococcus neoformans , the role of histone methyltransferases (HKMTs), one of the main epigenetic regulators, has been poorly elucidated. Herein, we characterize the role of a putative HKMT, Set202, in C. neoformans’ transition from the environment to the host. This is utmost important because this fungus’s main driver of disease is its ability to adapt to host conditions. We show that Set202 regulates a variety of processes needed for disease by acting as a true HKMT. Given the inadequate treatments available for diseases caused by this fungus, and that HKMTs are conserved in other fungal pathogens, characterizing Set202 function will provide crucial information to develop new treatments for this and other fungal diseases.

Catheter-associated urinary tract infections (CAUTIs) pose severe clinical challenges, often leading to urosepsis and multidrug resistance. The wound-healing process triggered by catheter-induced bladder damage deposits fibrinogen and its polymerized form, fibrin. However, uropathogens exploit the fibrinogen and fibrin matrix to form a protective biofilm. While catheter-induced inflammation recruits innate immune cells, particularly macrophages, to the site of infection, the persistence of uropathogens suggests that the local bladder environment alters their antimicrobial function. Our research pinpoints the coagulation cascade as a key driver of this dysfunction, finding a differential macrophage polarization influenced by fibrinogen and fibrin. We showed that urinary catheterization creates a fibrin-rich matrix that was correlated with polarizing macrophages into an anti-inflammatory M2-like state, suppressing their bactericidal response. In contrast, using mice expressing fibrinogen locked in the form of a monomer, we demonstrated that monomeric fibrinogen promotes a pro-inflammatory M1-like polarization state. Notably, skewing a M1-like macrophage state with GM-CSF failed to clear the infection and systemic dissemination, suggesting a dominant role for fibrin matrix in suppressing M1-like macrophage-mediated antimicrobial activity. Together, these findings show that the catheterized bladder provides signals that significantly alter macrophage function, creating an ideal niche for pathogen persistence.

Urinary catheterization, a frequent procedure in hospitals, nursing homes, and other healthcare facilities, is a primary driver of nosocomial infections. The most common of these are catheter-associated urinary tract infections (CAUTIs), which often progress to severe complication, sepsis, and ultimately death. The fungus Candida albicans is a primary causative agent of CAUTIs; yet, its tissue-specific pathogenesis remains poorly understood, which complicates development of efficient treatments. While Efg1 is a known virulence driver in CAUTI, its specific downstream targets within the unique bladder environment have not been defined. Here, we identify and validate the EFG1 regulon that is active during conditions that mimic the human catheterized bladder and, additionally, confirm the regulon by transcriptional profiling of catheters retrieved from patients with C. albicans infection. We found that this urine-specific signature is highly conserved in clinical samples, with Efg1-dependent genes being among the most robustly induced transcripts during active human infection. Furthermore, we characterized two of these key factors, ECE1 and EED1 , validating their roles in infection both in vitro in human urine and in vivo using a CAUTI mouse model. Elucidating this tissue-specific regulon offers a strategic roadmap for the development of targeted therapies to mitigate these ever-increasing life-threatening fungal infections.

Candidozyma auris is a high-priority, emerging fungal pathogen frequently isolated from urine in healthcare settings. These isolates are often associated with indwelling urinary catheters, a primary risk factor for catheter-associated urinary tract infections (CAUTIs). Despite its clinical prevalence, the mechanisms of C. auris colonization and pathogenesis within the bladder remain poorly understood. In this study, we screened C. auris isolates from diverse clades using an in vitro biofilm model and in vivo murine models of uncomplicated UTI and CAUTI. While in vitro biofilm formation varied among isolates, the presence of a catheter in vivo significantly enhanced fungal burden in the bladder. Notably, one strain (B11103) caused rapid systemic dissemination and mortality. To address this, we evaluated a liquid-infused silicone (LIS) catheter coating, which has previously shown efficacy against other uropathogens. The LIS coating significantly reduced C. auris attachment in vitro and, crucially, mitigated fungal burden on both the catheter and bladder tissue in vivo across all tested strains. For the hypervirulent B11103 strain, LIS catheters also significantly reduced dissemination to the kidneys and bloodstream. Furthermore, cytokine analysis revealed that C. auris CAUTI upregulates IL-6, CSF3, and CXCL1; importantly, this damaging inflammatory response was also dampened by the LIS catheter. These findings demonstrate that catheterization potentiates C. auris pathogenicity and identify LIS catheters as a promising, antimicrobial-sparing strategy to prevent colonization, systemic spread, and inflammation during C. auris CAUTI. IMPORTANCE: This research addresses the critical public health challenge posed by the emergence of Candidozyma auris, elucidating its pathogenesis in the urinary tract, the second-most common yet understudied reservoir. Here, we find that C. auris exhibits plasticity in its ability to form biofilms in urine and cause uncomplicated urinary tract infections (UTIs) and catheter-associated UTIs (CAUTIs). Importantly, we show that our liquid-infused silicone (LIS) catheters effectively disrupt this cycle by reducing fungal burden, preventing systemic spread, and dampening the damaging host inflammatory response. This work establishes the urinary tract as a critical niche for systemic entry and provides a validated strategy for infection prevention. Urinary catheters make C. auris dangerous, but this liquid-infused silicone coating is fighting back.

Cryptococcus neoformans, the etiological agent of cryptococcal meningitis (CM) is a globally distributed environmental yeast that mainly causes infections in immunocompromised individuals. Particularly in low-resource countries, the mortality rate of CM can reach 81% and accounts for ~19% of HIV/AIDS-related deaths each year. In immunocompromised individuals, once inhaled, C. neoformans escapes from the lungs and disseminates with special predilection for the central nervous system (CNS). Once in the brain, C. neoformans interacts with microglia, the tissue-resident macrophages of the CNS. Previous studies indirectly showed that microglia are ineffective at controlling this fungal infection. The mechanisms underlying this fungal survival and proliferation within the CNS, however, remain unclear. In this study, we use and validate the C20 immortalized human microglia cell line to study cryptococcal-microglia interactions. We show that microglia have limited phagocytic activity that is specific to C. neoformans and partly dependent on cryptococcal antiphagocytic proteins that alter cell size and cell wall structure. We also show that human microglia respond to cryptococcal strains differently than peripheral macrophages. Further, we show that human microglia are ineffective at killing phagocytosed C. neoformans , and that this could be due to the ability of this yeast to disrupt phagosome maturation and induce phagosome membrane damage in these cells. These findings provide us with fundamental knowledge regarding cryptococcal pathogenesis in the CNS, specifically the insight into how C. neoformans is recognized by microglia under different conditions and demonstrate the usefulness of C20 cells to further study how this yeast survives and replicates within the CNS environment. IMPORTANCE: While Cryptococcus neoformans is acquired through inhalation, the fatal pathology of cryptococcal infection occurs when the yeast disseminates to the central nervous system (CNS) and causes cryptococcal meningitis. Microglia are the first immune cells that C. neoformans will encounter once it reaches the CNS, and they are the largest population of macrophages in the brain. While microglia are professional phagocytes, they are unable to control C. neoformans infection. The mechanisms behind uncontrolled growth of C. neoformans within the CNS remain understudied, partly due to incomplete knowledge regarding microglia-cryptococcal interactions. This study provides fundamental knowledge into these interactions and establishes a powerful model to specifically study how C. neoformans is recognized by microglia and how cryptococcal phagosomes mature in these phagocytes. This work opens new avenues of research to further our understanding of cryptococcal-host interactions, which can be leveraged to develop more effective therapeutics for cryptococcal meningitis.

Candidozyma auris is a high-priority, emerging fungal pathogen frequently isolated from urine in healthcare settings. These isolates are often associated with indwelling urinary catheters, a primary risk factor for catheter-associated urinary tract infections (CAUTIs). Despite its clinical prevalence, the mechanisms of C. auris colonization and pathogenesis within the bladder remain poorly understood. In this study, we screened C. auris isolates from diverse clades using an in vitro biofilm model and in vivo murine models of uncomplicated UTI and CAUTI. While in vitro biofilm formation varied among isolates, the presence of a catheter in vivo significantly enhanced fungal burden in the bladder. Notably, one strain (B11103) caused rapid systemic dissemination and mortality. To address this, we evaluated a liquid-infused silicone (LIS)-catheter coating, which has previously shown efficacy against other uropathogens. The LIS-coating significantly reduced C. auris attachment in vitro and, crucially, mitigated fungal burden on both the catheter and bladder tissue in vivo across all tested strains. For the hyper-virulent B11103 strain, LIS-catheters also significantly reduced dissemination to the kidneys and bloodstream. Furthermore, cytokine analysis revealed that C. auris CAUTI upregulates IL-6, CSF3, and CXCL1; notably, this damaging inflammatory response was dampened by the LIS-catheter. These findings demonstrate that catheterization potentiates C. auris pathogenicity and identify LIS-catheters as a promising, antimicrobial-sparing strategy to prevent colonization, systemic spread, and inflammation during C. auris CAUTI. IMPORTANCE: This research addresses the critical public health challenge posed by the emergence of Candidozyma auris, elucidating its pathogenesis in the urinary tract, the second most common yet understudied reservoir. Here, we find that C. auris exhibits plasticity in its ability to form biofilms in urine and cause uncomplicated UTIs and catheter-associated UTIs. Importantly, we show that our liquid-infused silicone (LIS)-catheters effectively disrupt this cycle by reducing fungal burden, preventing systemic spread, and dampening the damaging host inflammatory response. This work establishes the urinary tract as a critical niche for systemic entry and provides a validated strategy for infection prevention.

Functionally similar to a plant vacuole or a mammalian lysosome, the fungal vacuole plays a vital role in many cellular processes. Most studies of the vacuole have been performed in the nonpathogenic yeast Saccharomyces cerevisiae; however, the vacuole in pathogenic fungi has recently been implicated in host invasion in both plants and mammals, highlighting an important role for the vacuole in pathogenesis. Here, we report that deletion of C. neoformans vacuolar protein 8 (VAC8) results in fragmented vacuole morphology, impairment of vacuolar fusion, and inability to form titan cells. Additionally, absence of Vac8 results in defective growth at high temperature and in the presence of caffeine, suggesting a defect in cell wall signaling. Interestingly, despite aberrant vacuole morphology, vac8Δ is slightly more resistant to fluconazole treatment, and displays increased resistance to hydrogen peroxide, suggesting the irregular vacuole morphology does not impair vacuolar function. Like S. cerevisiae Vac8, C. neoformans Vac8 is comprised of armadillo repeat regions which form alpha helices that fold to form a superhelix, allowing for increased protein–protein interaction. Many of the known binding partners of S. cerevisiae Vac8 are not present in the C. neoformans genome, suggesting novel functions for Vac8 in this fungus. Notably, deletion of VAC8 affected some virulence traits, providing support for targeting the fungal vacuole as a potential therapeutic intervention.

Urinary catheterization, a common procedure in hospitals and nursing home facilities, is a primary driver of hospital-acquired infections (HAI). These devices frequently lead to catheter-associated urinary tract infections (CAUTIs), which often progress to severe complication, sepsis, and ultimately death. The fungus Candida albicans has emerged as the second most common causative agent of CAUTIs; yet, its pathogenesis is poorly understood, which complicates development of efficient treatments. Previously, we identified the transcription factor Efg1 as a critical virulence driver in C. albicans CAUTIs. However, its specific downstream targets within the unique bladder microenvironment remained unknown. This study identifies, for the first time, the complete Efg1 regulon that is active during growth in human urine. We confirmed the clinical relevance of this discovery, finding that many of these Efg1-regulated factors are present and significantly upregulated in catheter samples from patients with C. albicans infections. Furthermore, we characterized two of these key factors, ECE1 and EED1 , validating their roles both in vitro in urine conditions and in vivo using a CAUTI mouse model. Identifying the tissue-specific downstream targets of Efg1 elucidates the precise mechanism of fungal CAUTI. This knowledge provides a new roadmap for developing targeted therapeutics, offering vital antimicrobial-sparing strategies to combat these life-threatening infections. SIGNIFICANCE: Catheter-associated urinary tract infections (CAUTIs) are common hospital-acquired infections that can lead to severe complications and death. Although most are caused by bacteria, the fungus Candida albicans is an increasingly prevalent cause, yet the pathogenesis of fungal CAUTIs is poorly understood. Previous research identified Efg1 as necessary for CAUTI, and now this study defines the urine-specific Efg1 regulon, validating its clinical relevance in catheter samples from infected patients. We further assessed how key downstream factors, Ece1 and Eed1, contribute to bladder infection. This first report of the urine-specific EFG1 network provides new targets for diagnosing and treating these life-threatening infections.

Cryptococcus neoformans, the etiological agent of cryptococcal meningitis (CM), is a globally distributed environmental yeast that mainly causes infections in immunocompromised individuals. Particularly in low-resource countries, the mortality rate of CM can reach 81% and accounts for 19% of HIV/AIDS-related deaths each year. In immunocompromised individuals, once inhaled, C. neoformans escapes from the lungs and disseminates with special predilection for the central nervous system (CNS). Once in the brain, C. neoformans interacts with microglia, the tissue-resident macrophages of the CNS. Previous studies indirectly showed that microglia are ineffective at controlling this fungal infection. The mechanisms underlying this fungal survival and proliferation within the CNS, however, remain unclear. In this study, we use and validate the C20 immortalized human microglia cell line to study cryptococcal-microglia interactions. We show that microglia have limited phagocytic activity that is specific to C. neoformans and partly dependent on cryptococcal antiphagocytic proteins that alter cell size and cell wall structure. We also show human microglia respond to cryptococcal strains differently than peripheral macrophages. Further, we show that human microglia are ineffective at killing phagocytosed C. neoformans , and that this could be due to the ability of this yeast to disrupt phagosome maturation and induce phagosome membrane damage in these cells. These findings provide us with fundamental knowledge regarding cryptococcal pathogenesis in the CNS, specifically insight into how C. neoformans is recognized by microglia under different conditions and demonstrate the usefulness of C20 cells to further study how this yeast survives and replicates within the CNS environment. Importance: While Cryptococcus neoformans is acquired through inhalation, the fatal pathology of cryptococcal infection occurs when the yeast disseminates to the central nervous system (CNS) and causes cryptococcal meningitis. Microglia are the first immune cells that C. neoformans will encounter once it reaches the CNS, and they are the largest population of macrophages in the brain. While microglia are professional phagocytes, they are unable to control C. neoformans infection. The mechanisms behind uncontrolled growth of C. neoformans within the CNS remains understudied, partly due to a lack of knowledge regarding microglia-cryptococcal interactions. This study provides fundamental knowledge into these interactions and establishes a powerful model to specifically study how C. neoformans is recognized by microglia and how cryptococcal phagosomes mature in these phagocytes. This work opens new avenues of research to further our understanding of cryptococcal-host interactions which can be leveraged to develop more effective therapeutics for cryptococcal meningitis.

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for >150,000 deaths every year, with a mortality rate as high as 81%. This high medical burden is due, in part, to an incomplete understanding of its pathogenesis. In a previous study, we identified a cryptococcal atypical Adenosine triphosphate (ATP)-binding cassette (ABC) pleiotropic drug resistance (PDR) transporter, PDR6 , that affected antifungal resistance and host interactions. Here, we follow up on the role of PDR6 in cryptococcal virulence. In vivo , mice infected with the pdr6 Δ strain display altered symptomatology and disease progression. Specifically, we observed a significant increase in the innate immune cell populations in the pdr6 Δ-infected mice when compared with their WT-infected littermates. Furthermore, quantification of pulmonary cytokines/chemokines revealed a robust increase of pro-inflammatory cytokines in mice infected with the pdr6 Δ mutant strain. Despite the documented sensitivity of the pdr6 Δ strain to azole antifungal drugs, the treatment of pdr6 Δ-infected animals with antifungals did not affect survival; however, treatment with a corticosteroid significantly extended survival, highlighting the importance of a balanced/controlled host immune response. Results with mice that mount opposing immune responses support our hypothesis that the pdr6 Δ strain induces a hyper-inflammatory immune response and that the mice succumb to immune-dependent tissue damage rather than the fungal burden. This altered immune response is driven, in part, by changes in the mutant’s surface. Taken together, this study provides insights regarding cryptococcal pathogenesis and highlights additional functions of PDR-type ABC transporters in pathogenic fungi. IMPORTANCE: Yeasts of the Cryptococcus genus, especially C. neoformans , can cause disease with unacceptably high mortality. This is due to delays in diagnostics, ineffective treatments, and an incomplete understanding of the interactions between this fungus and our immune system. In this study, we expand our knowledge of the biological function of the PDR6 gene, particularly its effect on modulating the host’s immune response. Normally, C. neoformans infections are characterized by an anti-inflammatory response that is unable to control the yeast. In the absence of PDR6 , the response to the infection is a dysregulated pro-inflammatory response that initially controls the fungi but eventually results in the death of the host due to too much tissue damage. This is due, in part, to an altered fungal surface. Given the dual role of PDR6 in modulating antifungal sensitivity and immune responses, this work provides important insights that may lead to new or improved therapeutics.