Biology subjects

Klingelhutz, A. J.

Publications and source records attributed to Klingelhutz, A. J..

8 recordsLinked to original sources

bioRxiv2025

Ebola's Hidden Target: Virus Transmission to and Accumulation within Skin

Ebola virus (EBOV), the causative agent of Ebola virus disease (EVD), remains one of WHOs top ten threats to global health. Infectious EBOV virions can be found on the surface of skin late during systemic infection and passed from the deceased through skin-to-skin contact. Here, we assess viral load and antigen expression in the skin of EBOV-infected non-human primates (NHP) and mouse adapted-EBOV (ma-EBOV) - infected mice and use the low containment viral model, rVSV/EBOV GP, to mechanistically define skin infection in mice. Viral RNA peaked within the skin proximal to the site of injection in EBOV-infected NHPs on day 6. In contrast, mouse skin sites distal to the site of ma-EBOV injection achieved maximal viral loads by day 3. At late times of infection, viral antigen-positive cells co-localized with markers for endothelial, stromal, and immune cells in the dermis. Epidermal cells within and surrounding hair follicles also harbored viral antigen, suggesting a potential mechanism of virus trafficking to the epidermal surface. Despite robust viral infection, distal skin sites of ma-EBOV-infected mice had low expression of proinflammatory stimulated genes. A similar cellular tropism was observed in the skin of mice infected with rVSV/EBOV GP, with discrete focal areas of intense infection. When virus was applied to the surface of gently abraded skin to remove the stratum corneum, epidermal keratinocytes were robustly infected, followed by systemic viral dissemination. To define cell surface receptors critical for virus trafficking to and replication within the skin, mice lacking the phosphatidylserine receptors were infected intraperitoneally with rVSV/EBOV GP. At day 3 of infection, skin distal to the site of infection of TIM-1 knock out (KO) mice had significantly lower levels of infectious virus than the control mice, suggesting that TIM-1 is essential for efficient distribution of virus to the skin. Our findings reveal that EBOV targets specific skin cell populations at late times of viral infection and that the host receptor TIM-1 is required for optimal viral dissemination.

microbiology↗

bioRxiv2024

Multiple dermal cell types support productive infection and dynamic translocation of infectious Ebola virus to the apical surface of human skin

Ebola virus (EBOV) within the Filoviridae family causes severe human disease. At late stages of infection, EBOV virions are found on the surface of patients skin; however, the permissive cell types within the skin and how infectious virus translocates to the apical skin surfaces is not known. Here, we describe a human transwell skin explant culture model and show that EBOV infection of human skin tissues via the basal media results in a time- and dose-dependent increase in infectious virus in dermal and epidermal tissue. Infectious virus was detected on the apical epidermal surface within 3 days, indicating that the virus propagates within and traffics through the tissue. In the dermis, EBOV-infected cells were of myeloid, endothelial and fibroblast origins, whereas keratinocytes harbored virus in the epidermis. Complementary studies showed that both purified skin fibroblasts and keratinocytes supported EBOV infection ex vivo and that both cell types required the phosphatidylserine receptor, Axl, and the endosomal protein, NPC1, for virus entry. Our experimental platform identified new susceptible cell types and demonstrated dynamic trafficking of EBOV virions that resulted in infectious virus on the skin surface; findings that may explain person-to-person transmission via skin contact. TeaserUsing a human skin explant model, these studies identify and characterize skin cell populations that support Ebola virus infection.

microbiology↗

bioRxiv2023

Disposition and Toxicity of 2,2',5,5'-Tetrachlorobiphenyl in Female Rats Following Intraperitoneal Exposure

The disposition and toxicity of lower chlorinated PCBs (LC-PCBs) with less than five chlorine substituents have received little attention. This study characterizes the distribution and metabolomic effects of PCB 52, an LC-PCB found in indoor and outdoor air, three weeks after intraperitoneal exposure of female Sprague Dawley rats to 0, 1, 10, or 100 mg/kg BW. PCB 52 exposure did not affect overall body weight. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis identified PCB 52 in all tissues investigated. Hydroxylated, sulfated, and methylated PCB metabolites, identified using GC-MS/MS and nontarget liquid chromatography-high resolution mass spectrometry (Nt-LCMS), were primarily found in the serum and liver of rats exposed to 100 mg/kg BW. Metabolomic analysis revealed minor effects on L-cysteine, glycine, cytosine, sphingosine, thymine, linoleic acid, orotic acid, L-histidine, and erythrose serum levels. Thus, the metabolism of PCB 52 and its effects on the metabolome must be considered in toxicity studies. HighlightsO_LIPCB 52 was present in adipose, brain, liver, and serum 3 weeks after PCB exposure C_LIO_LILiver and serum contained hydroxylated, sulfated, and methylated PCB 52 metabolites C_LIO_LIMetabolomics analysis revealed minor changes in endogenous serum metabolites C_LIO_LILevels of dopamine and its metabolites in the brain were not affected by PCB 52 C_LI

pharmacology and toxicology↗

bioRxiv2022

ISG15-modification of the Arp2/3 complex restricts pathogen spread

The ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogen-derived proteins. The consequences of ISG15 modification on substrate fate remain unknown. Here we reveal that ISGylation of the Arp2/3 complex slows actin filament formation and stabilizes Arp2/3 dependent structures including cortical actin and lamella. When properly controlled, this serves as an antibacterial and antiviral host defense strategy to directly restrict actin-mediated pathogen spread. However, Listeria monocytogenes takes advantage in models of dysregulated ISGylation, leading to increased mortality due to augmented spread. The underlying molecular mechanism responsible for the ISG15-dependent impact on actin-based motility is due to failed bacterial separation after division. This promotes spread by enabling the formation of multi-headed bacterial "bazookas" with stabilized comet tails that can disseminate deeper into tissues. A bacterial mutant that cannot recruit Arp2/3 or a non-ISGylatable mutant of Arp3 is sufficient to rescue slowed comet tail speed and restrict spread. Importantly, ISG15-deficient neonatal mice have aberrant epidermal epithelia characterized by keratinocytes with diffuse cortical actin, which could underlie observed defects in wound healing in human patients who lack ISG15. Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.

cell biology↗

bioRxiv2022

Native Adiponectin Plays a Role in the Adipocyte Mediated Conversion of Fibroblasts to Myofibroblasts

Adipocytes regulate tissues through production of adipokines that can act both locally and systemically. Adipocytes also have been found to play a critical role in regulating the healing process. To better understand this role, we developed a 3D human adipocyte spheroid system that has an adipokine profile similar to in vivo adipose tissues. Previously, we found that conditioned medium from these spheroids induces human dermal fibroblast conversion into highly contractile, collagen producing myofibroblasts through a transforming growth factor beta-1 (TGF-{beta}1) independent pathway. Here, we sought to identify how mature adipocytes signal to dermal fibroblasts through adipokines to induce myofibroblast conversion. By using molecular weight fractionation, heat inactivation, and lipid depletion, we determined mature adipocytes secrete a factor that is 30-100 kDa, heat labile, and lipid associated that induces myofibroblast conversion. We also show that depletion of the adipokine adiponectin, which fits those physiochemical parameters, eliminates the ability of adipocyte conditioned media to induce fibroblast to myofibroblast conversion. Interestingly, native adiponectin secreted by cultured adipocytes consistently elicited a stronger level of -SMA expression than exogenously added adiponectin. Thus, adiponectin secreted by mature adipocytes induces fibroblast to myofibroblast conversion and may lead to a phenotype of myofibroblasts distinct from TGF-{beta}1 induced myofibroblasts.

bioengineering↗

bioRxiv2022

Toxicity impacts on human adipose MSCs acutely exposed to Aroclor and non-Aroclor mixtures of PCBs.

PCBs accumulate in adipose where they may impact the growth and function of cells within the tissue. This is particularly concerning during adolescence when adipocytes expand rapidly. Herein we sought to understand how exposure to PCB mixtures found in U.S. schools affects human adipose mesenchymal stem/stromal cell (MSC) health and function. We investigated how exposure to Aroclor 1016 and Aroclor 1254, as well as a newly characterized non-Aroclor mixture that resembles the PCB profile found in cabinets, Cabinet Mixture, affects adipose MSC growth, viability, and function in vitro. We found that exposure to all three mixtures resulted in two distinct types of toxicity. At PCB concentrations >20 M, the majority of MSCs die, while at 1-10 M MSCs remained viable but display numerous alterations to their phenotype. At these sublethal concentrations, MSC rate of expansion slowed, and morphology changed. Further assessment revealed PCB-exposed MSCs had impaired adipogenesis and a modest decrease in immunosuppressive capabilities. Thus, exposure to PCB mixtures found in schools negatively impacts the health and function of adipose MSCs. This work has implications for human health due to MSCs role in supporting the growth and maintenance of adipose tissue. SYNOPSISPCB mixtures found in schools are toxic to human adipose mesenchymal stem/stromal cells, stunting their growth and altering their function in ways that could contribute to metabolic diseases.

pharmacology and toxicology↗

bioRxiv2022

Avian and Human Influenza Viruses Exhibit Distinct Glycoconjugate Receptor Specificities in Human Lung Cells

IAV utilize sialic acid (Sia) containing cell surface glycoconjugates for host cell infection, and IAV strains from different host species show preferences for structurally distinct Sia at the termini of glycoconjugates. Various types of cell surface glycoconjugates (N-glycans, O-glycans, glycolipids) display significant diversity in both structure and carbohydrate composition. To define the types of glycoconjugates that facilitate IAV infection, we utilized the CRISPR/Cas9 technique to truncate different types of glycoconjugates, either individually or in combination, by targeting glycosyltransferases essential to glycan biosynthesis in a human lung epithelial cell line. Our studies show that both human and avian IAV strains do not display strict preferences for a specific type of glycoconjugate. Interestingly, truncation of all three types of glycoconjugates significantly decreased the replication of human IAV strains, yet did not impact the replication of avian IAV strains. Taken together, our studies demonstrate that avian IAV strains utilize a broader repertoire of glycoconjugates for host cell infection as compared to human IAV strains. Author SummaryIt is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These glycoconjugates can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugates for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. Our studies indicate that avian IAV strains can utilize a wide variety of glycoconjugates for infection, whereas human IAV strains display restrictions in glycoconjugate type usage. These novel studies show distinct differences in host glycoconjugate preferences between human and avian IAV strains.

microbiology↗

bioRxiv2022

The Chlamydia trachomatis type III secreted effector protein CteG induces centrosome amplification through interactions with centrin-2

The centrosome is the main microtubule organizing center of the cell and is crucial for mitotic spindle assembly, chromosome segregation, and cell division. Centrosome duplication is tightly controlled, yet several pathogens, most notably oncogenic viruses, perturb this process leading to increased centrosome numbers. Infection by the obligate intracellular pathogen Chlamydia trachomatis (C.t.) correlates with blocked cytokinesis, supernumerary centrosomes, and multipolar spindles; however, the mechanisms behind how C.t. induces these cellular abnormalities from the confines of its inclusion, remain largely unknown. Here we show that the type III secreted effector protein, CteG, binds to centrin-2 (CETN2), a key structural component of centrosomes and regulator of centriole duplication. This interaction requires a functional calcium binding EF hand 4 of CETN2, which is recognized via the C-terminus of CteG. Significantly, we show that deletion of CteG, or knockdown of CETN2, significantly impairs chlamydias ability to induce centrosome amplification. Uniquely, we have identified the first bacterial effector to target centrins, crucial regulators of the eukaryotic cell cycle. These findings have not only allowed us to begin addressing how C.t. induces gross cellular abnormalities during infection, but also indicate that obligate intracellular bacteria may contribute to cellular transformation events that negatively impact host physiology even when the pathogen is long removed. Understanding the consequences of CteG-CETN2 interactions, its impact on centrosome amplification, and the long-term effect this has on host cells could explain why chlamydial infection leads to an increased risk of cervical or ovarian cancer. Significance StatementThe presence of more than two centrosomes is a hallmark of many types of cancer, including cervical and ovarian cancers of which Chlamydia trachomatis (C.t.) infection is a significant risk factor. Despite the importance of this problem, how C.t. orchestrates these drastic changes in the host cell remains poorly understood. Here, we describe how C.t. uses a single effector protein, CteG, to drive centrosome amplification via manipulation of a key regulator of centriole duplication, centrin-2. This work begins to define how C.t. induces centrosome amplification to promote its replication while potentially contributing to devastating long-term negative consequences for normal host physiology. Further it may help elucidate why chlamydial infection leads to an increased cancer risk.

microbiology↗