Biology subjects

Daniels, A. M.

Publications and source records attributed to Daniels, A. M..

5 recordsLinked to original sources

bioRxiv2026

Cryptosporidium sex employs a complex male fertilization apparatus that is required for continued infection

Most apicomplexan parasites replicate asexually to colonize their mammalian hosts, and sexually in the context of transmission. In contrast, for Cryptosporidium sex is essential to sustain intestinal infection in vivo and therefore is a promising opportunity to prevent and treat the disease. However, the mechanism of sex, which occurs within host enterocytes, is poorly understood, limiting the discovery of targets for such intervention. Here we identify an elaborate fertilization apparatus and numerous of its molecular components in male gametes. We discern two elements, one displayed on the apex, that makes intimate contact with host cells containing female parasites, and a second internal structure, that harbors the membrane fusogen HAP2. Ablation of HAP2 by conditional mutagenesis blocked fertilization and oocyst production, rapidly cured infected mice and left them with enhanced resistance to subsequent challenge. Our studies provide insights into a remarkable fertilization mechanism and highlight specific molecules as targets for translation.

microbiology↗

bioRxiv2026

Ablation of a maternal Cryptosporidium mRNA-binding protein results in sterile sporozoites

Infection with Cryptosporidium is a leading cause of diarrheal disease and early childhood mortality. This apicomplexan parasite undergoes asexual and sexual replication within the same host and recent studies have shown an intrinsic developmental program of obligate transition to male and female gametes and sex. While factors were identified that control male fate and development, how female gene expression is orchestrated remains largely unknown. Here we use the Cryptosporidium Single Cell Atlas to discover an RNA binding protein (F-RBP) as one of the earliest markers of female identity. Reporter parasites engineered based on this gene allowed us to calibrate transcriptional pseudotime against the real time of female development revealing a significant window of transcriptional fate ambiguity. While F-RBP is an early transcript, the protein persists throughout female development and into the zygote. Conditional ablation of the F-RBP gene showed it to be dispensable for sex determination and early female development in vitro. However, the gene is essential in vivo and its loss results in rapid cure. Cell biological experiments link this loss to the production of sterile oocysts which release sporozoites incapable of host cell invasion. F-RBP binds transcripts highly expressed in the female gamete enriched for a YBOX primary sequence motif and forms mRNA protein complexes in late females akin to processing or P bodies. We propose F-RBPs essential role to be in the regulation of long-term homeostasis of maternally inherited RNA required for sporozoite infectivity.

microbiology↗

bioRxiv2025

Harnessing droplet microfluidics and morphology-based deep learning for the label-free study of polymicrobial-phage interactions

Evaluating the impact of bacteriophages on bacterial communities is required to assess the future utility of phage therapy. Methods able to study bacterial polycultures in the presence of phages are useful to mimic evolutionary pressures found in natural environments and recapitulate complex ecological contexts. Bacteriophages can drive rapid genetic and phenotypic changes in host cells. However, the presence of other bacteria can also impact bacterial densities and community structure, and classical methods remain lengthy and resource intensive. Here, we introduce a microdroplet-based encapsulation method in which bacterial co-cultures are imaged using Z-stack brightfield microscopy. The method relies on automated droplet imaging using a novel AI-based autofocus function, coupled with morphology-based deep learning models for accurate identification of two morphologically distinct bacterial species. We show that we can monitor the relative growth dynamics of P. aeruginosa and S. aureus growing in 11 picolitre droplets for up to 24 hours. We demonstrate quantification of growth rates, bacterial densities and lysis dynamics of the two species without the need for plating. We show that a potent lytic phage of P. aeruginosa can either fully lyse the initial P. aeruginosa population or keep its density low long-term when in the presence of S. aureus.

microbiology↗

bioRxiv2024

Prodrug activation in malaria parasites mediated by an imported erythrocyte esterase, acylpeptide hydrolase (APEH)

The continued emergence of antimalarial drug resistance highlights the need to develop new antimalarial therapies. Unfortunately, new drug development is often hampered by poor drug-like properties of lead compounds. Prodrugging temporarily masks undesirable compound features, improving bioavailability and target penetration. We have found that lipophilic diester prodrugs of phosphonic acid antibiotics, such as fosmidomycin, exhibit significantly higher antimalarial potency than their parent compounds (1). However, the activating enzymes for these prodrugs were unknown. Here, we show that an erythrocyte enzyme, acylpeptide hydrolase (APEH) is the major activating enzyme of multiple lipophilic ester prodrugs. Surprisingly, this enzyme is taken up by the malaria parasite, Plasmodium falciparum, where it localizes to the parasite cytoplasm and retains enzymatic activity. Using a novel fluorogenic ester library, we characterize the structure activity relationship of APEH, and compare it to that of P. falciparum esterases. We show that parasite-internalized APEH plays an important role in the activation of substrates with branching at the alpha carbon, in keeping with its exopeptidase activity. Our findings highlight a novel mechanism for antimicrobial prodrug activation, relying on a host-derived enzyme to yield activation at a microbial target. Mutations in prodrug activating enzymes are a common mechanism for antimicrobial drug resistance (2-4). Leveraging an internalized host enzyme would circumvent this, enabling the design of prodrugs with higher barriers to drug resistance. SignificanceRising antimalarial drug resistance threatens current gains in malaria control. New antimalarial drugs are urgently needed. Unfortunately, many drug candidates have poor drug-like properties, such as poor absorbability in the gastrointestinal tract, or poor accumulation at the site of action. This can be overcome by prodrugging, the addition of prodrug groups which mask poor drug features until they are removed by an activating enzyme. Here, we show that a red blood cell enzyme, acylpeptide hydrolase, is taken up by malaria parasites and serves as the activating enzyme for multiple lipophilic ester prodrugs. Our findings highlight a novel mechanism for prodrug activation, which could be leveraged to design novel prodrugs with high barriers to drug resistance.

microbiology↗

bioRxiv2022

Adhesion of Crithidia fasciculata promotes a rapid change in developmental fate driven by cAMP signaling

Kinetoplastids are single-celled parasites responsible for human and animal disease. For the vast majority of kinetoplastids, colonization of an insect host is required for transmission. Stable attachment to insect tissues via the single flagellum coincides with differentiation and morphological changes. Although this process is essential for the generation of infectious forms, the molecular mechanism driving differentiation following adherence is not well understood. To study this process, we elaborate upon an in vitro model in which flagellated swimming cells of the kinetoplastid Crithidia fasciculata rapidly differentiate following adhesion to artificial substrates. Manipulation of culture parameters revealed that growth phase and time had a strong influence on the proportion of adherent cells. Live imaging of cells transitioning from swimming to an attached cell fate show parasites undergoing a defined sequence of events including an initial adhesion near the base of the flagellum, immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like structure between the tip of the shortened flagellum and the substrate. We have also assayed the role of the cyclic AMP (cAMP) signaling pathway in differentiation of C. fasciculata. Pharmacological inhibition of cAMP phosphodiesterases eliminated the ability of swimming cells to attach without affecting their growth rate. Further, treatment with inhibitor did not affect the growth rate of established attached cells, indicating its effect is limited to a critical window of time during the early stages of adhesion. Finally, in swimming parasites we have shown that a receptor adenylate cyclase localizes to the distal portion of the flagellum. In attached cells it is absent from the shortened flagellum and instead localizes to the cell body. Similarly, a putative phosphodiesterase, found along the length of the flagellum, also relocalizes to the cell body in attached parasites. These data suggest that in C. fasciculata cAMP signaling is required for adherence, that cAMP flux in the flagellum of swimming cells is spatially restricted, and that signaling domains may be reorganized during differentiation and attachment. These studies contribute to our understanding of the flagellum as a multi-functional organelle integrating processes related to motility, signaling, attachment, and differentiation and further develop C. fasciculata as a model kinetoplastid. Author SummaryParasites from the order Kinetoplastida are transmitted by insects and cause diseases such as Leishmaniasis, Chagas disease, and Human African Trypanosomiasis. A key aspect of the life cycle of these parasites is their ability to adhere to surfaces within the insect and subsequently differentiate into forms that are transmissable to their next host. Here, we explore the molecular mechanisms underpinning both adherence and differentiation using the mosquito parasite Crithidia fasciculata. This organism is closely related to pathogenic species but grows to high densities in culture and adheres robustly in vitro. We identified factors affecting the rate of adherence and defined morphological stages of this process including rapid shortening of the cells single flagellum. Using electron microscopy, we confirmed that adhesion to artificial substrates results in the formation of a filament-rich adhesive plaque at the tip of the flagellum that resembles the attachment to insect tissue. We also demonstrate the key role played by the cyclic AMP signaling pathway during adherence and differentiation. Specifically, pharmacological inhibition of enzymes that degrade cyclic AMP completely blocks adherence, and tagging of several proteins in this pathway show that their localization along the flagellum changes following differentiation to the attached form. These data provide insights into processes critical for all kinetoplastid life cycles.

microbiology↗