Tales from the Crypto: Phagolysosomal Phenomena Featuring Fungi

Host-pathogen interactions are a cornerstone of microbiology and medicinal research. Many incredible cellular mechanisms evolved from arms races between pathogens and host defenses. Studying these mechanisms leads to significant advances in molecular biology (ex. CRISPR/Cas) and medicine (ex. Penicillin). By understanding the delicate balance of the damage-response framework of microbial disease and studying how microbes and host cells outmaneuver each other, we can better understand pathogenesis to develop therapeutic strategies against debilitating disease. I study the host-pathogen interactions of Cryptococcus neoformans and macrophages, sites of various cellular phenomena with far reaching implications in fields ranging from immunology to bioremediation. I begin by describing phagolysosome acidification dynamics as a bet hedging strategy which macrophages employ to maximize fitness considering the variety of encounterable pathogens. Understanding this initial, broadly effective defense gives a frame of reference for downstream host-pathogen interactions. Phagolysosomal pH is an important aspect of this initial defense but is largely overlooked and undervalued. Next, I focus on the mechanism of C. neoformans macrophage-to-macrophage transfer. The existence of this phenomenon has been previously reported, but its mechanism is undiscovered. Using fluorescent microscopy with reporters specific to cellular compartments, antibody blockades for surface receptors, and live cell microscopy to follow infection outcomes I focus on understanding the circumstances of this phenomenon and place its mechanism within known cellular processes. Finally, I present work in progress toward understanding the interplay of C. neoformans and macrophages in the context of Dragotcytosis. Using transcriptomics, fluorescence microscopy, and simulations I outline how phagolysosome acidification and Dragotcytosis are linked and provide evidence suggesting Dragotcytosis is a survival mechanism to escape hostile phagolysosomes. These data bring new understanding to several facets of C. neoformans pathogenesis and suggest mechanisms which may be common to other human pathogens. Written under the advisory of Arturo Casadevall, Monica Mugnier, Dennis Wirtz, and Valeria Culotta

Estimating the size of fields in biomedical sciences

ABSTRACTScientific research output has increased exponentially over the past few decades, but not equally across all fields of study, and we lack clear methods for estimating the size of any given field of research. Understanding how fields grow, change, and are organized is essential to understanding how human resources are allocated to the investigation of scientific problems. In this study, we estimated the size of certain biomedical fields from the number of unique author names appearing in field-relevant publications in the PubMed database. Focusing on microbiology, where the size of fields is often associated with those who work on a particular microbe, we find large differences in the size of its subfields. We found that plotting the number of unique investigators as a function of time can show changes consistent with growing or shrinking fields. In general, the number of unique author names associated with a particular microbe correlated with the number of disease cases attributed to that microbe, suggesting that the microbiology field workforce is deployed in a manner consistent with the medical importance of the microbe in question. We propose that unique author counts can be used to measure the size of the workforce in any given field, analyze the overlap of the workforce between fields, and compare how the workforce correlates to available research funds and the public health burden of a field.IMPORTANCEScience and its individual fields are growing at spectacular rates along with the number of papers being generated each year. However, we lack methods to investigate the size of these fields, many times relying on anecdotal knowledge on which fields are “hot topics” or oversaturated. Thus, we developed a bibliometric method analyzing authorship information from PubMed to estimate the size of fields based on unique author counts. Our major findings are that unique author counts serve as an efficient measurement of the size of a given field. Additionally, the size of a biomedical science field correlates to its public health burden when compared to case numbers. This method allows us to compare growth rates, workforce distribution, and the allocation of resources between fields to understand how scientific fields self-regulate. These insights can, in turn, help guide policymaking, for example, in funding allocation, to ensure fields are not neglected

The capsule of Cryptococcus neoformans

The capsule of Cryptococcus neoformans is its dominant virulence factor and plays a key role in the biology of this fungus. In this essay, we focus on the capsule as a cellular structure and note the limitations inherent in the current methodologies available for its study. Given that no single method can provide the structure of the capsule, our notions of what is the cryptococcal capsule must be arrived at by synthesizing information gathered from very different methodological approaches including microscopy, polysaccharide chemistry and physical chemistry of macromolecules. The emerging picture is one of a carefully regulated dynamic structure that is constantly rearranged as a response to environmental stimulation and cellular replication. In the environment, the capsule protects the fungus against desiccation and phagocytic predators. In animal hosts the capsule functions in both offensive and defensive modes, such that it interferes with immune responses while providing the fungal cell with a defensive shield that is both antiphagocytic and capable of absorbing microbicidal oxidative bursts from phagocytic cells. Finally, we delineate a set of unsolved problems in the cryptococcal capsule field that could provide fertile ground for future investigations

Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms.

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment