Biology subjects

Smith, D. F. Q.

Publications and source records attributed to Smith, D. F. Q..

7 recordsLinked to original sources

bioRxiv2025

The Dynamics of Cryptococcus neoformans infection in Galleria mellonella

Galleria mellonella has emerged as an important host for the study of fungal virulence, insect immune responses, and the evaluation of antifungal agents. In this study we investigated the dynamics of fungal infections in G. mellonella using Cryptococcus neoformans, a human pathogenic fungus. Since the analysis of infection dynamics requires a fine temporal resolution of larval death, we employed a photographic timelapse technique that allowed us to simultaneously measure death by proxy of larval melanization and absence of movement. Larval mortality occurred in two phases, early and late, which differed in their timing of melanization. Early phase deaths occurred with rapid whole-body onset of melanization, followed by sudden cessation of movement several hours later. Contrastingly, late phase deaths occurred with a gradual cessation of movement, followed by melanization, typically radiating from one location on the larva. The differences in mortality kinetics suggests differences in fungal pathogenesis with one population succumbing early while the rest linger for later death. Subsequent analysis of mortality data using the inversion method revealed predictable deterministic dynamics without evidence for chaotic signatures, indicating that this C. neoformans-G. mellonella infection model behaves differently than bacterial-insect models. ImportanceThe ability to predict the course of an infection is critical to anticipating disease progression and effectively treating patients. Similarly, the ability to make predictions about pathogenesis in laboratory infection models could further our understanding of pathogenesis and lead to new treatments. As fungal diseases are expected to rise, understanding the dynamics of fungal infections will be important to anticipate and mitigate future threats. Here, we developed a timelapse method to visualize infections of Galleria mellonella larvae with the fungal pathogen Cryptococcus neoformans. This method provided insight into infection progression that are not apparent from standard survival measurement protocols, including the relationship between melanization and death. Further, it enabled us to explore the dynamics of disease progression in this system, which revealed deterministic dynamics without evidence of chaos, implying predictability in the outcome of cryptococcal infection in this moth.

microbiology↗

bioRxiv2024

Dietary L-3,4-dihydroxyphenylalanine (L-DOPA) augments cuticular melanization in Anopheles mosquitos while reducing their lifespan and malaria parasite burden

L-3,4-dihydroxyphenylalanine (L-DOPA), a naturally occurring tyrosine derivative, is prevalent in environments that include mosquito habitats, potentially serving as part of their diet. Given its role as a precursor for melanin synthesis we investigate the effect of dietary L-DOPA on mosquito physiology and immunity to Plasmodium falciparum and Cryptococcus neoformans infection. Dietary L-DOPA is incorporated into mosquito melanin via a non-canonical pathway and has a profound transcriptional effect associated with enhanced immunity, increased pigmentation, and reduced lifespan. Increased melanization results in an enhanced capacity to absorb electromagnetic radiation that affects mosquito temperatures. Bacteria in the mosquito microbiome act as sources of dopamine, a substrate for melanization. Our results illustrate how an environmentally abundant amino acid analogue can affect mosquito physiology and suggest its potential usefulness as an environmentally friendly vector control agent to reduce malaria transmission, warranting further research and field studies.

microbiology↗

bioRxiv2023

Thermal and pigment characterization of environmental fungi in the urban heat island of Baltimore City

A major barrier for most fungal species to infect humans is their inability to grow at body temperature (37{degrees}C). Global warming and more frequent extreme heat events may impose selection pressures that allow fungal adaptation to higher temperatures. Cities are heat islands that are up to 8{degrees}C warmer than their suburban counterparts because of mechanical heat production and reduced greenspace, among other factors, and may be an important reservoir of fungi that have increased risk of thermotolerance and inhabit environments near humans. Here we describe a novel and inexpensive technique that was developed to collect fungal samples from various sites in Baltimore, Maryland using commercially available taffy candy. Our results show fungal isolates from warmer neighborhoods show greater thermotolerance and lighter pigmentation relative to isolates of the same species from cooler neighborhoods, suggesting local adaptation. Lighter pigmentation in fungal isolates from warmer areas is consistent with known mechanisms of pigment regulation that modulate fungal cell temperature. The opportunistic pathogen Rhodotorula mucilaginosa from warmer neighborhoods had a higher resistance to gradual exposure to extreme heat than those from cooler neighborhoods. Our results imply fungal adaptation to increased temperature in an urban environment. The acquisition of thermotolerance poses a risk for humans if fungal species with pathogenic potential acquire the capacity to grow at human body temperatures and cause disease.

microbiology↗

bioRxiv2022

Melanization of Candida auris is Associated with Alteration of Extracellular pH

Candida auris is a recently emerged global fungal pathogen, which causes life-threatening infections, often in healthcare settings. C. auris infections are worrisome because the fungus is often resistant to multiple antifungal drug classes. Furthermore, C. auris forms durable and difficult to remove biofilms. Due to the relatively recent, resilient, and resistant nature of C. auris, we investigated whether it produces the common fungal virulence factor melanin. Melanin is a black-brown pigment typically produced following enzymatic oxidation of aromatic precursors, which promotes fungal virulence through oxidative stress resistance, mammalian immune response evasion, and antifungal peptide and pharmaceutical inactivation. We found that certain strains of C. auris oxidized L-DOPA and catecholamines into melanin. Melanization occurred extracellularly in a process mediated by alkalinization of the extracellular environment, resulting in granule-like structures that adhere to the fungus external surface. C. auris had relatively high cell surface hydrophobicity, but there was no correlation between hydrophobicity and melanization. Melanin protected the fungus from oxidative damage, but we did not observe a protective role during infection of macrophages or Galleria mellonella larvae. In summary, C. auris alkalinizes the extracellular medium, which promotes the non-enzymatic oxidation of L-DOPA to melanin that attaches to its surface, thus illustrating a novel mechanism for fungal melanization.

microbiology↗

bioRxiv2022

Melanization is an important antifungal defense mechanism in Galleria mellonella hosts

A key component of insect immunity is melanin encapsulation of microbes. Melanization is also a part of an immune process known as nodulation, which occurs when insect hemocytes surround microbes and produce melanin. Insect nodules are analogous to mammalian immune granulomas. Melanin is believed to kill microbes through the production of toxic intermediates and oxidative damage. However, it is unclear to what extent immune melanin is directly fungicidal during infections of insect hosts. We reported previously that C. neoformans cells are encapsulated with host-derived melanin within hemocyte nodules. Here we report an association between melanin-based immune responses by Galleria mellonella wax moth larvae and fungal cell death of C. neoformans during infection. To monitor melanization in situ, we applied a tissue-clearing technique to G. mellonella larvae, revealing that nodulation occurs throughout the organism. Further, we developed a protocol for time-lapse microscopy of extracted hemolymph following exposure to fungal cells, which allowed us to visualize and quantify the kinetics of the melanin-based immune response. Using this technique, we found evidence that cryptococcal melanins and laccase enhance immune melanization in hemolymph. We used these techniques to also study the fungal pathogen Candida albicans infections of G. mellonella. We find that the yeast form of C. albicans was the primary targets of host melanization, while filamentous structures were melanin-evasive. Approximately 23% of melanin-encapsulated C. albicans yeast survive and break through the encapsulation. Overall, our results provide direct evidence that the melanization reaction functions as a direct antifungal mechanism in insect hosts.

microbiology↗

bioRxiv2022

On the relationship between Pathogenic Potential and Infective Inoculum

Pathogenic Potential (PP) is a mathematical description of an individual microbe, virus, or parasites ability to cause disease in a host, given the variables of inoculum, symptomology, mortality, and in some instances, median survival time of the host. We investigated the relationship between pathogenic potential (PP) and infective inoculum (I) using two pathogenic fungi in the wax moth Galleria mellonella with mortality as the relevant outcome. Our analysis for C. neoformans infection revealed negative exponential relationship between PP and I. Plotting the log(I) versus the Fraction symptomatic (Fs) over median host survival time (T) revealed a linear relationship, with a slope that varied between the different fungi studied and a y-intercept corresponding to the inoculum that produced no symptoms. The I vs Fs/T slope provided a measure of the pathogenicity of each microbial species, which we call the pathogenicity constant or kPath. The kPath provides a new parameter to quantitatively compare the relative virulence and pathogenicity of microbial species for a given host. In addition, we investigated the PP and Fs/T from values found in preexisting literature. Overall, the relationship between Fs/T and PP versus inoculum varied among microbial species and extrapolation to zero symptoms allowed the calculation of the lowest pathogenic inoculum (LPI) of a microbe. Microbes tended to fall into two groups: those with positive linear relationships between PP and Fs/T vs I, and those that had a negative exponential PP vs I relationship with a positive logarithmic Fs/T vs I relationship. The microbes with linear relationships tended to be bacteria, whereas the exponential-based relationships tended to be fungi or higher order eukaryotes. Differences in the type and sign of the PP vs I and Fs/T vs I relationships for pathogenic microbes suggest fundamental differences in host-microbe interactions leading to disease. Author SummaryThe ability of a microbe, virus, or parasite to cause disease is dependent on multiple factors, virulence factors. host immune defenses, the infective inoculum, and the type of immune response. For many microbes their capacity for causing disease is highly dependent on the inoculum. The mathematical formula for Pathogenic Potential (PP) is a way to compare the ability of an organism to have a pathogenic effect, as measured by Fraction Symptomatic (Fs), mortality (M), and inoculum (I), and can include the median survival time of the host (T). Increasing inoculum of the fungus Cryptococcus neoformans for a moth host resulted in exponentially smaller pathogenic potential, and the Fs/T versus inoculum plot showed a logarithmic relationship. Together, these relationships show diminishing returns with increasing cryptococcal inoculum, in which each individual fungus plays a smaller role in pathogenicity. Literature data shows that other microbes, mostly bacteria, had linear Fs/T versus inoculum relationships, which indicate that each bacterium contributed an equal amount to pathogenicity. These differences in relationships can point to differences in host-microbe interactions and suggest new ways in which the organism causes disease.

microbiology↗

bioRxiv2020

Glyphosate Inhibits Melanization and Increases Insect Susceptibility to Infection

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including: UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanins broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate - the most widely used herbicide globally - inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in two evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria- causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosates mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance Overall, these findings suggest that glyphosates environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

microbiology↗