Mike Reichelt - Academia.edu (original) (raw)
Papers by Mike Reichelt
Virology, Mar 1, 2011
The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investig... more The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investigate the characteristics and functions of the ORF11 gene product. ORF11 is expressed as a 118kDa polypeptide in VZV-infected cells; the protein is present in the nucleus and cytoplasm and is incorporated into VZ virions. Although ORF11 had little effect in transactivating VZV gene promoters in transfection assays, deleting ORF11 from the virus was associated with reduced expression of immediate early proteins IE4, IE62 and IE63, and the major glycoprotein, gE. ORF11 was identified as an RNA binding protein and its RNA binding domain was defined. However, disrupting the ORF11 RNA binding domain did not affect skin infection, indicating that RNA binding capacity, conserved among the alphaherpesviruses homologues, is not essential while the contribution of ORF11 to the expression of the IE proteins and gE may be required for VZV pathogenesis in skin in vivo.
Microscopy and Microanalysis, 2010
Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, ... more Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.
Microscopy and Microanalysis
Molecular Therapy - Nucleic Acids
Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) s... more Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase–endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)—a highly aggressive malignancy with a dismal posttreatment prognosis—implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy—previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed subst...
<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h a... more <p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s006" target="_blank">Video S6</a>. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s007" target="_blank">Video S7</a>. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s008" target="_blank">Video S8</a>.</p
Proceedings of the National Academy of Sciences
Acinetobacter baumannii is a clinically important, predominantly health care–associated gram-nega... more Acinetobacter baumannii is a clinically important, predominantly health care–associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii , we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL i...
The thyroid functions at the apex of a web of endocrine organs that control cell growth, differen... more The thyroid functions at the apex of a web of endocrine organs that control cell growth, differentiation and metabolic homeostasis. Thyroid dysregulation significantly impacts human health in myriad ways with thyroid diseases standing as the most common endocrine disorder. Despite the essential role of the thyroid in human health, a high-resolution view of the cellular composition as well as molecular mechanisms that govern function of this crucial organ have been lacking. Employing the first single-cell analyses of adult mouse thyroid, we here report the discovery of unexpected thyrocyte heterogeneity, specifically three distinct thyrocyte subtypes marked by different metabolic and Notch signaling patterns. Using a battery of pharmacologic and genetic methods, we find that selective inhibition of Notch ligands and receptors disrupts thyrocyte mitochondrial activity and ROS production, thus decreasing levels of circulating thyroid hormones, inducing hypothyroidism and disrupting who...
Microscopy and Microanalysis
<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h a... more <p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) BSE-SEM images at different magnifications of a syncytium of VZV infected melanoma cells. Left panel: low magnification view of a syncytium; middle panel: one nucleus of the same syncytium with two PML cages; right panel: higher magnification view of a PML cage with sequestered VZV capsids. Black squares indicate areas that are shown at higher magnification in the panels to the right. Scale bars are 5 µm. (B) Five representative images (s1, s5, s9, s13, s17) from a series of 18 consecutive sections through the nucleus shown in A, middle panel. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s004" target="_blank">Video S4</a>. (C and D) 3D models based on tracing and segmentation in all 18 sections of electron dense heterochromatin (blue); nucleocapsids (yellow spheres) and PML cages (green, shown only in D). 1,732 and 1,324 capsids were identified in the upper and lower PML cage, respectively). Scale bars are 5 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s005" target="_blank">Video S5</a>. (E and F) 3D models of the upper PML cage. Color code as above, but immature capsids (A and B-type capsids) are shown as yellow spheres and mature capsids (C-type) in orange; the PML cage is transparent green (shown only in F). Scale bars are 2 µm.</p
<p>Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial... more <p>Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s001" target="_blank">Video S1</a>. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s003" target="_blank">Video S3</a>. All scale bars are 5 µm.</p
<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h a... more <p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s010" target="_blank">Video S10</a>. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s011" target="_blank">Video S11</a>. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).</p
Journal of Bacteriology, 2021
As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classe... more As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp .
Investigative Ophthalmology & Visual Science, 2016
Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, ... more Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. Disruption of the physiological barriers that prevent these organisms from disseminating outside their assigned niches leads to inflammatory responses. Using segmented filamentous bacteria (SFB) as a sentinel species, we demonstrate that the IL-23/IL-22 pathway dynamically and selectively modulates the relative abundance of certain commensals in the adult animal. Genetic or pharmacological inactivation of the pathway results in defective barrier function, leading to systemic microbial dissemination and enhanced TH17 responses in distal tissues. Furthermore, we demonstrate suppression of TH17 responses in wild-type animals upon activation of the IL-22 pathway. Our data therefore suggests that IL-23/IL-22 pathway is of critical importance for normal immune homeostasis through the maintenance of mucosal barrier function.
SSRN Electronic Journal, 2020
Acinetobacter baumannii is an extracellular Gram-negative human pathogen that causes life-threate... more Acinetobacter baumannii is an extracellular Gram-negative human pathogen that causes life-threatening infections but the virulence mechanisms it employs remain poorly understood. Here we report that A. baumannii secretes outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) that are targeted to host cell mitochondria thereby inducing DRP1-driven mitochondrial fragmentation, ROS production and cell death. DRP1 loss reverses these phenotypes. Using intra-nasal lung infection model in mice, we show that OmpAAb is required for bacteria-induced damage to alveolar macrophages and for bacterial dissemination from the lung to other organs. Heterologous expression of OmpAAb in E. coli transfers its virulence properties to E. coli and is sufficient to induce mitochondrial fragmentation and cell death in infected cells. We describe the mechanism by which extracellular A. baumannii utilizes OmpAAb to inflict host cell damage and promotes virulence in-vitro and in-vivo and further report the host cell factor responsible for the effect.
Microscopy and Microanalysis, 2018
Scientific Reports, 2018
A correction to this article has been published and is linked from the HTML and PDF versions of t... more A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
Journal of Histochemistry & Cytochemistry, 2019
Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic... more Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workf...
Virology, Mar 1, 2011
The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investig... more The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investigate the characteristics and functions of the ORF11 gene product. ORF11 is expressed as a 118kDa polypeptide in VZV-infected cells; the protein is present in the nucleus and cytoplasm and is incorporated into VZ virions. Although ORF11 had little effect in transactivating VZV gene promoters in transfection assays, deleting ORF11 from the virus was associated with reduced expression of immediate early proteins IE4, IE62 and IE63, and the major glycoprotein, gE. ORF11 was identified as an RNA binding protein and its RNA binding domain was defined. However, disrupting the ORF11 RNA binding domain did not affect skin infection, indicating that RNA binding capacity, conserved among the alphaherpesviruses homologues, is not essential while the contribution of ORF11 to the expression of the IE proteins and gE may be required for VZV pathogenesis in skin in vivo.
Microscopy and Microanalysis, 2010
Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, ... more Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.
Microscopy and Microanalysis
Molecular Therapy - Nucleic Acids
Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) s... more Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase–endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)—a highly aggressive malignancy with a dismal posttreatment prognosis—implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy—previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed subst...
<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h a... more <p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s006" target="_blank">Video S6</a>. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s007" target="_blank">Video S7</a>. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s008" target="_blank">Video S8</a>.</p
Proceedings of the National Academy of Sciences
Acinetobacter baumannii is a clinically important, predominantly health care–associated gram-nega... more Acinetobacter baumannii is a clinically important, predominantly health care–associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii , we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL i...
The thyroid functions at the apex of a web of endocrine organs that control cell growth, differen... more The thyroid functions at the apex of a web of endocrine organs that control cell growth, differentiation and metabolic homeostasis. Thyroid dysregulation significantly impacts human health in myriad ways with thyroid diseases standing as the most common endocrine disorder. Despite the essential role of the thyroid in human health, a high-resolution view of the cellular composition as well as molecular mechanisms that govern function of this crucial organ have been lacking. Employing the first single-cell analyses of adult mouse thyroid, we here report the discovery of unexpected thyrocyte heterogeneity, specifically three distinct thyrocyte subtypes marked by different metabolic and Notch signaling patterns. Using a battery of pharmacologic and genetic methods, we find that selective inhibition of Notch ligands and receptors disrupts thyrocyte mitochondrial activity and ROS production, thus decreasing levels of circulating thyroid hormones, inducing hypothyroidism and disrupting who...
Microscopy and Microanalysis
<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h a... more <p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) BSE-SEM images at different magnifications of a syncytium of VZV infected melanoma cells. Left panel: low magnification view of a syncytium; middle panel: one nucleus of the same syncytium with two PML cages; right panel: higher magnification view of a PML cage with sequestered VZV capsids. Black squares indicate areas that are shown at higher magnification in the panels to the right. Scale bars are 5 µm. (B) Five representative images (s1, s5, s9, s13, s17) from a series of 18 consecutive sections through the nucleus shown in A, middle panel. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s004" target="_blank">Video S4</a>. (C and D) 3D models based on tracing and segmentation in all 18 sections of electron dense heterochromatin (blue); nucleocapsids (yellow spheres) and PML cages (green, shown only in D). 1,732 and 1,324 capsids were identified in the upper and lower PML cage, respectively). Scale bars are 5 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s005" target="_blank">Video S5</a>. (E and F) 3D models of the upper PML cage. Color code as above, but immature capsids (A and B-type capsids) are shown as yellow spheres and mature capsids (C-type) in orange; the PML cage is transparent green (shown only in F). Scale bars are 2 µm.</p
<p>Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial... more <p>Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s001" target="_blank">Video S1</a>. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s003" target="_blank">Video S3</a>. All scale bars are 5 µm.</p
<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h a... more <p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s010" target="_blank">Video S10</a>. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s011" target="_blank">Video S11</a>. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).</p
Journal of Bacteriology, 2021
As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classe... more As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp .
Investigative Ophthalmology & Visual Science, 2016
Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, ... more Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. Disruption of the physiological barriers that prevent these organisms from disseminating outside their assigned niches leads to inflammatory responses. Using segmented filamentous bacteria (SFB) as a sentinel species, we demonstrate that the IL-23/IL-22 pathway dynamically and selectively modulates the relative abundance of certain commensals in the adult animal. Genetic or pharmacological inactivation of the pathway results in defective barrier function, leading to systemic microbial dissemination and enhanced TH17 responses in distal tissues. Furthermore, we demonstrate suppression of TH17 responses in wild-type animals upon activation of the IL-22 pathway. Our data therefore suggests that IL-23/IL-22 pathway is of critical importance for normal immune homeostasis through the maintenance of mucosal barrier function.
SSRN Electronic Journal, 2020
Acinetobacter baumannii is an extracellular Gram-negative human pathogen that causes life-threate... more Acinetobacter baumannii is an extracellular Gram-negative human pathogen that causes life-threatening infections but the virulence mechanisms it employs remain poorly understood. Here we report that A. baumannii secretes outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) that are targeted to host cell mitochondria thereby inducing DRP1-driven mitochondrial fragmentation, ROS production and cell death. DRP1 loss reverses these phenotypes. Using intra-nasal lung infection model in mice, we show that OmpAAb is required for bacteria-induced damage to alveolar macrophages and for bacterial dissemination from the lung to other organs. Heterologous expression of OmpAAb in E. coli transfers its virulence properties to E. coli and is sufficient to induce mitochondrial fragmentation and cell death in infected cells. We describe the mechanism by which extracellular A. baumannii utilizes OmpAAb to inflict host cell damage and promotes virulence in-vitro and in-vivo and further report the host cell factor responsible for the effect.
Microscopy and Microanalysis, 2018
Scientific Reports, 2018
A correction to this article has been published and is linked from the HTML and PDF versions of t... more A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
Journal of Histochemistry & Cytochemistry, 2019
Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic... more Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workf...