Biology subjects

Wallbank, B. A.

Publications and source records attributed to Wallbank, B. A..

7 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↗

bioRxiv2025

Diverse infections transcriptionally reprogram the intestinal epithelium and epithelial-immune cell interactions

The distal small intestine plays vital roles in host physiology by regulating nutrient and fluid homeostasis. Despite being impacted in Crohns disease and a major target for a range of infections, we know relatively little about the complexity of cellular responses and cell-cell communication in the ileum during infection. Single cell and spatial transcriptomics have emerged as powerful technologies to study tissue heterogeneity in the gut, but these tools have focused on the large intestine, in part due to the accessibility of this tissue for biopsies and its importance in cancer. Here we present GutPath, an atlas of over 500,000 single cells with RNA and protein expression profiles for 91 cell states in the ileum across diverse infectious archetypes. We show that GutPath accurately captures established immune responses to infection while revealing pathogen-specific responses in enterocytes. To highlight the discovery potential of this atlas, we identify a novel enterocyte cell state present during Yersinia pseudotuberculosis infection that is spatially linked to bacterial load and tissue pathology. GutPath establishes a much-needed resource for the immunology community that will accelerate the study of the transcriptional diversity of cellular landscapes in the small intestine.

immunology↗

bioRxiv2023

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-gamma on parasite restriction

The production of IFN-{gamma} is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. The use of single cell RNA sequencing to profile IEC during infection revealed induction of IFN-{gamma}-dependent gene signatures that was comparable between uninfected and infected cells, and IEC expression of the IFN-{gamma} receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-{gamma} demonstrated the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-{gamma} on IEC and suggest a model in which IFN-{gamma}-mediated bystander activation of uninfected enterocytes is important for control of Cryptosporidium. AUTHOR SUMMARYThe cytokine interferon-gamma (IFN-{gamma}) plays an important role in the control of intracellular infections by a wide variety of bacteria, viruses and parasites. While the impact of IFN-{gamma} on immune cells has been a major research focus, how it impacts intestinal epithelial cells remains poorly understood. Cryptosporidium parasites are an important cause of morbidity in a variety of epidemiological settings and exclusively infect intestinal epithelial cells (IEC). Recent advances in the ability to genetically modify and study Cryptosporidium in wild-type hosts provides a useful model to investigate IEC-intrinsic mechanisms of pathogen control. In this study, single cell RNA-sequencing was used to analyze the IEC response to infection and IFN-{gamma} signalling. We demonstrate broad changes in the epithelial compartment during infection that include the induction of an IEC population with robust induction of IFN-{gamma}-stimulated genes. In addition, we show that infected IEC remain responsive to IFN-{gamma} signalling, and that this cytokine causes a delayed reduction in parasite burden that correlates with the kinetics of IEC responsiveness to IFN-{gamma} stimulation. Together, our work uncovers how Cryptosporidium infection impacts the IEC compartment and helps define the relationship between the kinetics of IFN-{gamma} responsiveness and pathogen control in IEC.

immunology↗

bioRxiv2023

Intestinal cDC1s provide IL-12 dependent and independent functions required for CD4+ T cell mediated resistance to Cryptosporidium

Cryptosporidium is an enteric pathogen that is a prominent cause of diarrheal disease. Control of this infection requires CD4+ T cells, though the processes that lead to T cell-mediated resistance have been difficult to assess. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to dissect the early events that influence CD4+ T cell priming and effector function. These studies highlight that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node (mesLN) and differentiate into Th1 cells in the gut, where they mediate IFN-{gamma}-dependent control of the infection. Although type 1 conventional dendritic cells (cDC1s) were not required for initial priming of CD4+ T cells, cDC1s were required for CD4+ T cell expansion and gut homing. cDC1s were also a major source of IL-12 that was not required for priming but promoted full differentiation of CD4+ T cells and local production of IFN-{gamma}. Together, these studies reveal distinct roles for cDC1s in shaping CD4+ T cell responses to enteric infection: first to drive early expansion in the mesLN and second to drive effector responses in the gut. SummaryCryptosporidium parasites that express model antigens were generated to dissect how parasite-specific CD4+ T cells are primed and mediate effector functions required to control this enteric pathogen. cDC1s produced IL-12p40 and were required for early expansion and gut homing of CD4+ T cells. However, IL-12p40 was only required for the development of Th1 CD4+ T cell effector function in the gut.

immunology↗

bioRxiv2023

Dendritic cell-mediated responses to secreted Cryptosporidium effectors are required for parasite-specific CD8+ T cell responses

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the MHC-I restricted SIINFEKL epitope which is recognized by TCR transgenic OT-I CD8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of IFN-{gamma} that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (ROP1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells (IEC), type I dendritic cells (cDC1) were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as targets of the immune system and suggest that crosstalk between enterocytes and cDC1s is crucial for CD8+ T cell responses to Cryptosporidium.

immunology↗

bioRxiv2019

Global profiling of myristoylation in Toxoplasma gondii reveals key roles for lipidation in CDPK1 and MIC7 function

N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase has been proposed as an attractive drug target in several pathogens. Functionally the myristate often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence also suggests additional regulatory roles for myristoylation on proteins. Here we describe the first global chemoproteomic screening of protein myristoylation in Toxoplasma gondii. Through quantitative mass spectrometry coupled with validated chemoproteomic tools, we identify 65 myristoylated proteins. We report functionally important myristoylation on the key signalling protein CDPK1 and, surprisingly, myristoylation of the microneme protein 7 (MIC7), a predicted type-I-transmembrane protein. We demonstrate that myristoylation of MIC7 is not important for the trafficking to micronemes, but appears to play a role in host cell invasion. This dataset represents a large fraction of the parasites myristoylated proteome and a prerequisite to investigate this modification in Toxoplasma.

microbiology↗

bioRxiv2018

Phosphorylation of a Toxoplasma gondii tyrosine transporter by calcium-dependent kinase 3 is important for parasite fitness

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. The calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout ({Delta}cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with {Delta}cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, a putative transporter of the major facilitator superfamily (MFS) and show that it is rapidly phosphorylated after induction of calcium signalling. Conditional knockout (KO) of the transporter reveals an essential role in the lytic cycle during intracellular growth with a transcriptome signature of amino acid-starved parasites. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import. Complementation with phosphorylation site mutants shows that phosphorylation of serine 56 (S56) by TgCDPK3 gives the parasites a growth benefit in competition assays. Collectively, these findings validate an important, albeit non-essential role for TgCDPK3 in the regulation of metabolic processes, in addition to motility.\n\nAuthor summaryToxoplasma gondii is an obligate intracellular parasite. To survive and spread throughout the host it must repeatedly infect, replicate within and exit, host cells. These recurring cycles of infection and egress rely on signalling pathways that allow the parasites to sense and respond rapidly to their environment. While some key kinases and secondary messengers within these pathways have been identified, functional analysis of non-kinases has been very limited. This is especially true for candidates that are not predicted to play a role in active motility or are not known to function in established signalling pathways. Here we have followed up on an unexpected target of the T. gondii calcium-dependent kinase 3 (TgCDPK3), a plant-like calcium dependent kinase, that was previously shown to play an important role in calcium-mediated exit from the host cell. We show that, in addition to controlling motility of the parasite (as previously shown), TgCDPK3 phosphorylates an essential tyrosine transporter in the plasma membrane. Mutational analysis of the phosphorylation sites demonstrates an important role in maintaining parasite fitness, thus demonstrating that TgCDPK3 plays a pleiotropic role in controlling both egress and metabolism.

microbiology↗