Projects
Pathogenesis and immune response to fungal pathogens
Host response to Cryptococcus sp.
We use real-time live-cell microscopy to explore the interactions during early infection of macrophages with different strains of C. neoformans and C. gattii.
Virulence factors of Cryptococcus sp.
Cryptococcal cells expresses a suite of virulence mechanisms that contribute to their ability to survive ingestion by host macrophages. These include a polysaccharide capsule, melanin pigment layer, urease-catalyzed ammonia production, and phospholipase activity. Research in our lab is aimed at characterizing the individual factors and how they sometimes interact during infection.
Primary metabolism and fungal virulence
Contact: Alberto Patiño (jpatino2@jh.edu)
Primary metabolism is essential for development. Nutrient availability conditions the metabolic state of the cell; consequently, other features, such as metabolic plasticity, stress response, and virulence potential during host interactions, among others, are also influenced. Energy production is based on two organelles: mitochondria and peroxisomes. In this project, we investigate the regulation between metabolism (with a focus on mitochondrial and peroxisomal activity) and fungal virulence.
Quorum sensing and C. neoformans virulence
Contact: Alberto Patiño (jpatino2@jh.edu)
Quorum sensing is a mechanism that coordinates population behavior and positively regulates virulence. In this project, we investigate the signaling and epigenetic regulation that mediate quorum sensing in C. neoformans.
Characterization of cryptococcal polysaccharides
Contact: Maggie Wear (mwear1@jhu.edu)
The key virulence factor of cryptococal pathogens Cryptococcus neoformans and Cryptococcus gattii are the polysaccharides they produce. These polysaccharides, both capsular polysaccharide (CPS) and shed or exopolysaccharide (EPS) are responsible for protecting fungal cells from dehydration and predation in nature and evading the immune system and macrophage phagocytosis during infection. While we have characterized functions for both EPS and CPS, our understanding of their synthesis, assembly, transport, structure, and genetic regulation all remain in their infancy. Current projects in this area utilize bioinformatics, biochemistry, and biophysics to further characterize the polysaccharides themselves (primary and secondary structure) as well as to identify the genes regulating repeat motif selection, synthesis, transport, and assembly of the capsule.
Cryptococcal polysaccharide conjugated vaccine
Contact: Piotr Stempinski (pstempi1@jhu.edu)
Cryptococcosis is a life-threatening fungal disease caused primarily by Cryptococcus neoformans, which disproportionately affects immunocompromised individuals. In this project, we developed an experimental vaccine targeting a key virulence factor of C. neoformans—the exopolysaccharide (EPS). By conjugating minimally processed EPS to the well-established protein carrier CRM197 and testing two adjuvants, we demonstrated that the vaccine could elicit strong antibody responses and improve survival in a mouse model of cryptococcosis. Mice vaccinated with the EPS-CRM197 conjugates showed enhanced immune recognition and protection against infection. We are now building on these promising results by optimizing the conjugation process, testing additional carrier proteins, and exploring new adjuvants to further improve vaccine efficacy. Through these continued efforts, we aim to understand how each component contributes to immune protection and to develop a robust, broadly protective, and clinically viable vaccine against cryptococcosis.
The Cryptococcus spore surface and its role in host invasion
Contact: Sébastien Ortiz (sortiz11@jh.edu)
The Cryptococcus yeast morphotype has been widely studied, and the study of its anti-phagocytic capsule has been critical to combating this pathogen; however, the yeast cell type is not the only morphotype to consider in cryptococcal disease. Cryptococcus produces dormant and stress resistant basidiospores (sexual spores) that are smaller and better aerosolized than yeast and thus more likely to reach the lower airways. Importantly these spores have a distinct surface to yeast, lacking the anti-phagocytic capsule. Spores are a key presumed infectious morphotype, yet due to difficulties associated in working with spores, the Cryptococcus spore surface remains undefined and relatively few studies exist evaluating spore-host interactions. Critically, Cryptococcus spores can disseminate out of the host lung better than yeast which translates to spores of otherwise avirulent yeast causing disease in intranasal murine models of cryptococcosis. This preferential dissemination and disease are likely a result of spores being able to invade host lung cells better than yeast. As spores germinate into yeast, and become more yeast-like, this preferential internalization diminishes probably due to surface epitopes being masked as the yeast capsule is formed. The spore surface components that drive host cell invasion, dissemination and disease remain a mystery. Our research is centered on defining the distinct surface components of spores, determining which components drive preferential host cells invasion, and identifying the molecular mechanisms enabling Cryptococcus dissemination and disease.
Environmental transmission of Cryptococcus sp.
Contact: Isabel Jimenez (isabeljimenez@jhmi.edu)
Cryptococcal cells have various strategies to increase their buoyancy in water and thus facilitate their transport via aqueous routes. Prolonged buoyancy of fungal cells also suggests that aerosolization of water containing Cryptococcus could result in respiratory exposure in susceptible hosts. In this project, we focus on characterizing mechanisms by which fungal cells can be transported, including wind, smoke, and water.
Investigating antibody-mediated pathogenesis of Candida auris
Contacts: Samuel Rodrigues (sroadri1@jh.edu)/Daniel Smith (dsmit274@jh.edu)
C. auris is a recently-emerged fungal pathogen that forms notoriously durable biofilms on surfaces and is often resistant to at least one class of existing antifungal medications used to treat infections. In this project, we will infect mice with C. auris and characterize the array of antibodies produced against the fungus. Developing and characterizing monoclonal antibodies against C. auris will allow us to understand whether antibody-mediated immunity provides any protection against C. auris infection, which antibodies have the strongest protective effect, and which can be used as potential therapeutics in future clinical trials.
Role of melanin in infectious disease
Characterization of fungal melanin
Melanin, a widespread, heteropolymeric pigment, contributes to cryptococcal virulence by impairing host immune cell-mediated killing and reducing susceptibility to antifungal drugs. Our research aims to elucidate the mechanisms underlying cryptococcal cell wall melanization, including the role of lipids in the process, and to identify key similarities and differences between fungal and mammalian melanosomes. We also investigate how melanin alters host-pathogen interactions and explore the potential of laccase, the enzyme that catalyzes the initial step in the melanin biosynthetic pathway, as a novel therapeutic target in the treatment of cryptococcosis.
Melanin in insect defenses
Contact: Emma Camacho (ecamach2@jhmi.edu)
Melanin is one of the most ancient and versatile pigments produced by most living organisms, including mosquitoes. Malaria-transmitting mosquitoes as well as other mosquito vectors are the deadliest animals on earth, causing devastating infectious diseases. In mosquitoes, melanin is essential for their survival at both aquatic and terrestrial stages. I am most interested in how mosquitoes adapt their melanin-based defense mechanisms in response to changes in the environment, and how their diets modulate gut microbiota-brain axis to impact mosquito’s ability to cause disease. My approach to these questions, leveraged on foundational knowledge in fungal melanin studies, integrates omics analyses, molecular biology, biochemistry, immunology, and ecological perspectives. Through this work, I hope to advance our understanding of the biological connections between melanin, malaria parasites, and microbiome dynamics shaping host defense and pathogen transmission.
Melanin masquerading yeast are sugar-hungry killers!
Contact Francisco Hernandez (fhernan8@jhu.edu)
Pigments, like melanins, are ubiquitous throughout biological kingdoms of fauna and flora, useful for predator evasion, reproduction, thermal regulation, attractants, energy production, and many more. Fungi have specifically primed melanin pigmentation as an evolutionary enhancement of virulence to outcompete the host’s defenses. This project explores novel roles of why melanin is so critical for establishing infection by inducing a primitive pathway that powers proliferation in the state of nutrient depletion. This project focuses on a key enzyme that flips a switch to unleash a sugar-crazed arms race.
Antibody-mediated catalysis and the immune response
The role of catalytic antibodies in response to infection
Contact Maggie Wear (mwear1@jhu.edu)
We have been characterizing antibody-mediated catalysis in cryptococcal infections and SARS-CoV-2 infections and observe that both fungal and viral infections result in the production of antibodies which are capable of cleaving their antigen. We are currently working to characterize the mechanism of action for this catalysis and have ruled out the known proteolytic mechanisms. Further work is needed to understand the mechanism of antibody-mediated catalysis and if these catalytic antibodies are also produced during bacterial infection.
Adenovirus vectored antibody delivery protects mice against Cryptococcus neoformans infection
Contact: Hannah Tsingine (htsing1@jhmi.edu)
Monoclonal antibodies are promising therapies for use in the treatment of fungal diseases. In the case of Cryptococcus neoformans, there is a large body of pre-clinical data showing that antibodies can protect against infection and synergize with antifungal drugs in animal models of experimental cryptococcosis. Two of the most studied monoclonal antibodies, 18B7 and 2H1, bind to the glucuronoxylomannan (GXM) polysaccharides of the capsule thus increasing opsonization and neutralization of the fungus by macrophages. The adeno-associated virus immunoprophylaxis (AAV VIP) system is a newly developed system designed to use cloning technology to develop protective chimeric antibodies containing the variable regions of 2H1 and/or 18B7. We have built a recombinant plasmid containing the variable light and heavy chains 2H1 and 18B7 and fused the variable regions with human IgG constant regions. The plasmids are then packaged in AAV capsules, and infection is delivered intramuscularly. Upon AAV infection, mice were found to produce chimeric antibodies at low expression and were well tolerated pre-challenge. These results establish the feasibility of using the new AAV system to induce antibody production in mice and suggest that it is a promising approach for the prevention and treatment of cryptococcosis.
Fungal evolution
Environmental preconditioning: what experiences make fungi better?
Contacts: Isabel Jimenez (isabeljimenez@jhmi.edu)/Daniel Smith (dsmit274@jh.edu)
Cryptococcus predominantly lives in the environment and spends most of its life cycle, and evolutionary time, in the soil. Prior studies show that interactions with amoeba in the soil can make cryptococcal cells more virulent towards mammalian cells. We hypothesize that the relationship between fungi and the environment is one in which environmental pressures - such as osmotic stress, pH, UV light, temperature, and more - lead to fungal evolution and thus, climate change and other environmental alterations can drive fungi to change in ways that may also be cross-protective once fungi encounter mammalian cells. In this project, we focus on understanding the pathways that C. neoformans and C. gattii use to resist environmental stressors, including temperature and osmotic stress, and how the environmental experiences of fungal cells can influence its later encounters with mammalian phagocytes.
Ancient fungi at the Cretaceous/Paleogene boundary
Contact: Rosanna Baker (rosanna@jhmi.edu)
Geological time is punctuated by major extinction events caused by global calamities and fungi, acting as essential microbial decomposers, often flourish in the aftermath of such catastrophes. Fossilized fungal and plant spores preserved in ancient sedimentary rocks provide valuable records of these ecological upheavals and shifts in their relative abundances can reveal periods of intensified fungal activity. We analyzed samples from three geological sites in North America and identified two fungal proliferative spikes dating to approximately 66 million years ago. The fungal blooms are coincident with two global cataclysms: massive Deccan volcanic eruptions near the end of the Cretaceous period and a large asteroid impact in the Yucatan Peninsula that is credited with causing extinction of all non-avian dinosaurs. By characterizing these ancient fungal communities, we aim to gain insight into the dynamics of this mass extinction and the role fungi may have played in shaping post-catastrophe ecosystem recovery.
Fungal polysaccharides: how are they related?
Contact: Maggie Wear (mwear1@jhu.edu)
While C. neoformans and C. gattii are the only known encapsulated fungi, there are a number of other fungal species that produce polysaccharides. The characterized cryptococcal polysaccharides are glucuronoxylomannan (GXM), and glucuronoxylomannogalactan (GXMgal). In the capsule of C. neoformans and C. gattii GXM is reported to make up 85-90% while GXMgal is reported at 4-7% with mannoproteins and other proteins making up the remaining material. Many of these non-cryptococcal fungal polysaccharides are bound by the cryptococcal anti-GXM mAb 18B7, earning them the title “GXM-like.” Beyond their ability to be bound by 18B7, the structural characterization of these GXM-like polysaccharides is not well known, however, they play important roles in biofilm formation, ROS defense, and pathogenesis.
Other microbiology projects
Characterizing the function of aquaporins in C. neoformans
Contact: Piotr Stempinski (pstempi1@jhu.edu)
In this study, we investigated the role of Aqp1, an aquaporin protein in the opportunistic fungal pathogen Cryptococcus neoformans, using a combination of phenotypic characterization, structural modeling, and predictive analyses. Our results demonstrate that Aqp1 regulates cell size and promotes titan-like cell formation in a pH-dependent manner under host-mimicking, capsule-inducing conditions. We also found that Aqp1 contributes to the management of intracellular reactive oxygen species, implicating it in the fungal oxidative stress response. These findings identify Aqp1 as a novel regulator of cryptococcal morphogenesis and highlight its role in environmental sensing and stress adaptation. Overall, our work provides new insights into the molecular mechanisms underlying titan cell development and expands our understanding of aquaporin function in fungal pathogenesis.
Astromycology lab
Contact: Radamés Cordero (rcorder4@jhu.edu)
Fungi are among the most resilient life forms on Earth, yet we know little about how they adapt and survive in the extreme conditions of space. Our laboratory investigates the physiological and structural responses of fungi to spaceflight stressors—including radiation, microgravity, and vacuum exposure. By uncovering how these organisms maintain viability and stability in such environments, we aim to advance understanding of microbial risks to astronaut health and to harness fungal biology for engineered living materials (ELMs) that protect and sustain life beyond Earth.
Microbial populations in cacao fermentation
Contact: Maggie Wear (mwear1@jhu.edu)
Cacao is grown mostly in south american and african countries, however, Hawaii is an ideal climate for cacao growth and production. In my work with Lydgate Farms on the island of Kauai we have been studying the microbes involved in cacao fermentation to determine (1) the ideal microbial inoculum for successful fermentation (2) the origin of the microbes that contribute to different phases of fermentation, and (3) to determine which microbes contribute small molecule flavinoids important for chocolate flavor.
Investigating an innovative strategy to preserve Anopheles mosquito eggs
Contact: Emma Camacho (ecamach2@jhmi.edu)
Drawing on mechanistic insights from our fungal cell wall melanization studies, this project aims to investigate the innovative potential of a sugar meal supplemented with L-DOPA and N-acetylglucosamine, which serve as precursors for melanin and chitin, respectively, to promote oviposition of desiccation-resistant eggs in female Anopheles mosquitoes. Through a comprehensive experimental design, this study will assess the synergistic effects of this combined diet on embryonic development as a low-cost, sustainable solution that overcomes the associated challenges of generating mosquito transgenic lines.