Biology subjects

Denton, S. L.

Publications and source records attributed to Denton, S. L..

7 recordsLinked to original sources

bioRxiv2026

Recombinant Ehrlichia TRP75 Rescues Molting Failure of Ticks Infected with TRP75 Mutant Ehrlichia

Ehrlichia species are obligatory intracellular bacteria that exist in nature through a cycle of infection and transmission between ticks and their mammalian hosts, potentially causing a severe febrile illness called ehrlichiosis in humans and animals. Roles of Ehrlichia molecules in tick-mammal Ehrlichia transmission are mostly unknown. Tandem repeat protein (TRP) 75 is a lipoprotein conserved among Ehrlichia species. We identified and cloned a Himar1 transposon insertional mutant of Ehrlichia japonica that lacks C-terminal intrinsically disordered region of TRP75 (TRP75{Delta}C). This mutant was competent in infecting and replicating in both mammalian and tick cells in culture, and causing fatal illness similar to wild-type (WT) E. japonica. Upon feeding on TRP75{Delta}C and WT Ehrlichia-infected mice, both larval- and nymphal-stage of Ixodes scapularis ticks ingested similar amounts of blood meal as those fed on uninfected mice, and acquired similar levels of infection. However, most of TRP75{Delta}C Ehrlichia-infected ticks failed to complete molting, in contrast to successful molting of WT Ehrlichia-infected and uninfected ticks by 10 weeks post-feeding. In both WT Ehrlichia-infected and uninfected ticks, ~1,000-fold exponential increase in ecdysone receptor (EcR) mRNA occurred at 15 days post-feeding, whereas EcR mRNA was not upregulated in TRP75{Delta}C Ehrlichia-infected ticks. Upon microinjection of recombinant full-length TRP75 protein into engorged ticks infected with TRP75{Delta}C Ehrlichia, molting efficiency was ~90% restored, and there was significantly increased EcR mRNA compared with solvent microinjection control. This suggests that the endoparasitic bacterial molecule TRP75 manipulates the molting success of infected ticks and EcR mRNA levels for the survival of bacteria-hosted ticks and transstadial transmission.

microbiology↗

bioRxiv2026

Anaplasma phagocytophilum Subversion of Host Hepcidin-Ferroportin Iron Nutritional Immunity

Obligatory intracellular bacterium Anaplasma phagocytophilum (Aph) causes an emerging infectious disease called human granulocytic anaplasmosis. Within host cells, Aph proliferates in a membrane-bound vacuole (Aph-vacuole), to which all required nutrients must be routed, including essential iron. Here, using a fluorescent labile iron-binding dye, we found that Aph-vacuoles are enriched with labile iron. Further, ferroportin (Fpn), the only known iron exporter in the plasma membrane of host cells, was increasingly localized to Aph-vacuole post-invasion. Plasma membrane Fpn is tightly regulated by the hormone hepcidin, which binds to Fpn and triggers its ubiquitination and internalization. We found Fpn-GFP mutants deficient in ubiquitination (domain deletion or Y64H) had reduced or absent localization to Aph-vacuoles. Fpn-GFP (D131V) and Fpn-GFP (D039A) mutants that are deficient in iron binding and transport could still localize to Aph-vacuoles, but significantly reduced Aph growth and labile iron in Aph-vacuoles. Aph infection upregulated host cell expression of the hepcidin mRNA and protein. Furthermore, the gene encoding hepcidin is upregulated by inflammation, and we found Aph induced strong IL-6, IL-1{beta}, and TNF- mRNA expression in the host cells. Altogether, these results suggest that Aph induces production of proinflammatory cytokines for autocrine or paracrine hepcidin secretion to trigger the Fpn internalization, which can then be diverted to Aph-vacuoles in a ubiquitination-dependent manner as a mechanism of iron acquisition for thebacteria. This finding illuminates pathogen manipulation of cellular iron export mechanisms, and subversion of host hepcidin-Fpn iron nutritional immunity against pathogens. IMPORTANCEIron is an essential element for both humans and microorganisms, serving as a cofactor in key metabolic processes. Obligatory intracellular bacterium Anaplasma phagocytophilum infects and proliferates within a membrane-bound vacuole (Aph-vacuoles) of neutrophils and endothelial cells, competitively acquiring intracellular iron from the host. Upon exploration of cytoplasmic labile free iron levels and distribution in the A. phagocytophilum-infected and uninfected host cells, we uncovered the unique ability of this bacterium to enrich intravacuolar iron. Ferroportin is the only cellular iron exporter of the host cells, which is regulated by the hepatic hormone hepcidin. Herein, we investigate the role of ferroportin and endogenous hepcidin locally produced by the host cells for iron enrichment in Aph-vacuoles. Ultimately, this study provides new insights into the novel mechanisms of microbial manipulation of host cells to acquire the essential micronutrient iron and overcoming host nutritional immunity, which may facilitate more effective treatment and prevention.

molecular biology↗

bioRxiv2024

CD4 Co-Receptor Regulates Sex-Specific NK Cell Responses to Acute Toxoplasma gondii Infection.

Immunity to Toxoplasma gondii (T. gondii) is sexually dimorphic in humans and mice, with females having higher morbidity and mortality during immune dysfunction and HIV-AIDS. The mechanisms underlying these sex differences are unclear. We investigated how a lack of CD4+ T cells (CD4 co-receptor KO) impacted T. gondii survival in mice. Female CD4 co-receptor KO mice succumbed to T. gondii much faster than males. To dissect why female CD4 co-receptor KO mice died faster, we tested their NK cell responses to acute T. gondii infection compared to males. Although in wild-type (WT) animals, both sexes had similar increases in total NK cells and IFN{gamma} + NK cells, infected CD4 co-receptor KO female mice had 50% fewer IFN{gamma}+ NK cells than infected WT female mice. Infected male CD4 co-receptor KO had a similar increase in IFN{gamma}+ NK cells as WT male mice. Since CD4 co-receptor deficient mice still have functional helper T cells that are CD4-, we next tested survival and NK cell responses in female and male MHCII deficient (MHCIIKO) animals, which completely lack helper CD4+T cells. Surprisingly, survival, NK cell numbers, and IFN{gamma}+ NK cells were not significantly different between WT or MHCIIKO female and male mice. These results suggest CD4 co-receptor expression is required for survival via optimal NK cell responses during acute T. gondii infection only in female mice and not in male mice. Our findings reveal an unappreciated sexual dimorphic role of CD4 co-receptor expression in regulating NK cell responses to acute T. gondii infection.

immunology↗

bioRxiv2023

Transferrin Receptor 1 is Required for CD4+ T Cell Iron-Dependent Response During Acute Toxoplasma gondii Infection

Elemental iron is an essential nutrient involved in many biological processes including infection and immunity. How iron impacts Toxoplasma gondii (T. gondii) in vivo and development of immunity during infection is unclear. We found that although iron is required for parasite proliferation in vitro, paradoxically, iron restriction in vivo increased parasite burdens during acute and persistent infection stages and decreased survival of mice. Iron restriction lowered IL-12 and IFN{gamma} in spleen and serum, but ratios of myeloid cells and the number and function of Natural Killer cells were unchanged. Iron restriction significantly impaired the development of CD4+ and CD8+ T cell responses to T. gondii during replicating type II and attenuated vaccine strain cps1-1 infection. Low iron conditions reduced the percent and absolute numbers of antigen experienced CD11a+CD49d+, functional IFN{gamma}+, and CD62L-KLRG1+ effector T cells. Iron restriction also decreased vaccine efficacy of cps1-1 strain against secondary lethal challenge. Antigen experienced CD4+ and CD8+ T cells both upregulated their iron transporter Transferrin receptor 1 (CD71) during infection regardless of iron restriction. Mice whose CD4+ T cells were deficient in CD71 had reduced CD4+ T cell antigen experience and polyfunctionality, yet CD8+ T cell responses remained intact, and their long term survival was not affected compared to wild type litter mate controls. This study highlights that iron acquisition by T cells is required for activation and vaccine induced long-term protection against T. gondii. Understanding how iron affects multiple immune compartments will be essential to define iron regulation of immunity to T. gondii.

immunology↗

bioRxiv2023

Theft of Host Transferrin Receptor-1 by Toxoplasma gondii is required for infection

Nutrient acquisition by apicomplexan parasites is essential to drive their intracellular replication, yet the mechanisms that underpin essential nutrient acquisition are not defined. Using the apicomplexan model Toxoplasma gondii, we show that host cell proteins including the transferrin receptor 1, transferrin, ferritin heavy and light chains, and clathrin light chain are robustly taken up by tachyzoites. Tachyzoite acquisition of host cell protein was not related to host cell type or parasite virulence phenotypes. Bradyzoites possessed little capacity to acquire host cell proteins consistent with the cyst wall representing a barrier to host cell protein cargo. Increased trafficking of host cell transferrin receptor 1 and transferrin to endolysosomes boosted tachyzoite acquisition of host proteins and growth rate. Theft of host transferrin 1 and transferrin did not significantly affect iron levels in the tachyzoite. This study provides insight into essential functions associated with parasite theft of host iron sequestration and storage proteins.

microbiology↗

bioRxiv2019

NK cells negatively regulate CD8 T cells to promote immune exhaustion and chronic Toxoplasma gondii infection

NK cells regulate CD4+ and CD8+ T cells in acute viral infection, vaccination and the tumor microenvironment. NK cells also become exhausted in chronic activation settings. The mechanisms causing these ILC responses and their impact on adaptive immunity are unclear. CD8+ T cell exhaustion develops during chronic Toxoplasma gondii (T. gondii) infection resulting in parasite reactivation and death. How chronic T. gondii infection impacts the NK cell compartment is not known. We demonstrate that NK cells do not exhibit hallmarks of exhaustion. Their numbers are stable and they do not express high PD1 or LAG3. NK cell depletion with anti-NK1.1 is therapeutic and rescues chronic T. gondii infected mice from CD8+ T cell exhaustion dependent death, increases survival after lethal secondary challenge and reduces parasite reactivation. Anti-NK1.1 treatment increased polyfunctional CD8+ T cell responses in spleen and brain and reduced CD8+ T cell apoptosis. Chronic T. gondii infection promotes the development of a modified NK cell compartment, which does not exhibit normal NK cell behavior. This splenic CD49a-CD49b+NKp46+ NK cell population develops during the early chronic phase of infection and increases through the late chronic phase of infection. They are Ly49 and TRAIL negative and are enriched for expression of CD94/NKG2A and KLRG1. They do not produce IFN{gamma}, are IL-10 negative, do not increase PDL1 expression, but do increase CD107a on their surface. They are also absent from brain. Based on the NK cell receptor phenotype we observed NKp46 and CD94-NKG2A cognate ligands were measured. Activating NKp46 (NCR1-ligand) ligand increased and NKG2A ligand Qa-1b expression was reduced. Blockade of NKp46 also rescued the chronically infected mice from death. Immunization with a single dose non-persistent 100% protective T. gondii vaccination did not induce this cell population in the spleen, suggesting persistent infection is essential for their development. We hypothesize chronic T. gondii infection induces an NKp46 dependent modified NK cell population that reduces functional CD8+ T cells to promote persistent parasite infection in the brain. NK cell targeted therapies could enhance immunity in people with chronic infections, chronic inflammation and cancer.

immunology↗

bioRxiv2019

Anti-Asialo GM1 treatment during secondary Toxoplasma gondii infection is lethal and depletes T cells

Using vaccine challenge model of T. gondii infection, we found that treatments with two commonly used for NK cell depletion antibodies resulted in different survival outcomes during secondary infection. Anti-ASGM1 resulted in 100% death and greater parasite burden at the site of infection than anti-NK1.1. Anti-NK1.1 treatment resulted in increased parasite burdens, but animals did not die. Further we found that anti-ASGM1 treatment depleted T cells. CD8+ T cells were more susceptible that CD4+ T cells to the treatment. ASGM1 was expressed on a higher percentage of CD8+ T cells than CD4+ T cells and CD8+ T cells. In T. gondii-immunized animals ASGM1 was enriched on effector memory (Tem) and central memory (Tcm) CD8+ T cells. However, Tem were more susceptible to the treatment. After secondary infection, Tem, Tcm, effector (Tef) and naive (Tn) CD8+ T cells were positive for ASGM1. Anti-ASGM1 treatment during reinfection resulted in greater depletion of activated IFN{gamma}+, Granzyme B+, Tem and Tef than Tcm and Tn CD8+ T cells. Anti-ASGM1 also depleted IFN{gamma}+ CD4+ T cells. Recombinant IFN{gamma} supplementation prolonged survival of anti-ASGM1 treated mice, demonstrating that this antibody eliminated IFN{gamma}-producing T and NK cells important for control of the parasite. These results highlight that anti-ASGM1 antibody is not an optimal choice for targeting only NK cells and more precise approaches should be used. This study uncovers ASGM1 as a marker of activated effector T cells and the potential importance of changes in sialylation in lipid rafts for T cell activation during T. gondii infection.

immunology↗