Journal: bioRxiv

Article Title: Dynamic Reprogramming of Fungal Cell Walls Underlies Germination and Immune Exposure in Zygomycetous Fungal Pathogens

doi: 10.64898/2026.05.12.724644

Figure Lengend Snippet: ( A ) TEM images of the dormant conidia reveal a multilayered cell wall architecture, including an electron-dense outer layer and inner polysaccharide-rich regions. ( B ) At high magnification, osmium-fixed, epoxy-embedded samples (left) preserve structural integrity, whereas LR White–embedded samples prepared without OsO 4 (right) enhance contrast in the outer wall. Sub-labels denote (i) outer electron-dense region, (ii) granular region, and (iii) electron-lucent inner layer. Without OsO 4 , the outer layer appears as a thin electron-lucent band consistent with melanin, with the remaining regions largely electron-lucent. ( C ) Rigid molecules in the dormant conidial cell walls were detected by 2D 13 C- 13 C CORD spectra, showing signals from β-1,3-glucan (B), chitin (Ch), and chitosan (Cs). Superscripts indicate different structural forms of each polysaccharide, and the numbers denote carbon positions. For example, Cs a 1-2 represents the correlation between carbons 1 and 2 in type-a chitosan. ( D ) Structural representations of carbohydrates. Yellow bars indicate whether each molecule is detected in the rigid phase, the mobile phase, or in both. NMR abbreviations are given. Key carbon sites are numbered. ( E ) Composition of rigid carbohydrates in dormant conidia based on intensity analysis of the 2D CORD spectrum. ( F ) Overlay of 2D CORD spectra of dormant conidia (blue) and 3-day-old mycelia (orange). ( G ) Cytochemical labeling of dormant conidia shows gold-specific staining for β-1,3-glucan with Fc-Dectin-1, chitin (with Wheat germ agglutinin lectin (WGA-lectin), and anti-chitosan rabbit antiserum. Black dots represent electron-dense gold particles indicating probe binding. Scale bars: 500 nm. ( H ) Mobile molecules in dormant conidia detected by the 2D 13 C-DP refocused J-INADEQUATE spectrum. In addition to β-1,3-glucan and two forms of chitosan, signals are detected for Fuc (F), Gal, α-1,2-Man (Mn , ), and α-1,6-Man (Mn , ). ( I ) Cytochemical binding of Concanavalin-A lectin (ConA) to dormant conidial cell walls confirms the presence of mannans and/or glycoproteins. Zoomed-in view of 2D 13 C-DP J-INADEQUATE spectrum was presented for ( J ) C5-C6 correlations of polymeric fucoses and ( K ) glucuronic acid (GlcA). Polymeric fucoses show five forms (a,b, c, d, i) as traced by dashed lines in dormant conidia (blue) while type-e is present only in mycelia (orange).

Article Snippet: Grids were washed with endotoxin-free water and incubated for 1 h with 100 μg mL -1 recombinant human Fc-Dectin-1 (Fc-hDectin-1a, InvivoGen).

Techniques: Labeling, Staining, Binding Assay

Journal: bioRxiv

Article Title: Dynamic Reprogramming of Fungal Cell Walls Underlies Germination and Immune Exposure in Zygomycetous Fungal Pathogens

doi: 10.64898/2026.05.12.724644

Figure Lengend Snippet: ( A ) TEM images of germinating conidia showing swollen conidia undergoing isotropic expansion at 1 h (left), swollen conidia with rupture of the outer cell wall enabling emergence of a polarized germ tube (middle), and elongated germ tubes (right). Magenta arrows indicate changes in the outer wall, while yellow arrows highlight the thinner inner layer that extends and contributes to germ tube wall formation. ( B ) Violin plot showing the reduction in cell wall thickness from dormant conidia (DC) to swollen (SW) and germ tube (GT) stages (n=120 per group). ( C ) 1D 13 C CP spectra detecting only neosynthesized polysaccharides in germinating 12 C-conidia grown in 13 C-labeled media (inset). Spectra were collected at different time points of 1 h (black), 2.5 h (green), and 5 h (magenta). Dashed lines highlight the signature peaks of chitin, β-1,3-glucan, and chitosan. ( D ) Molar composition of rigid neo-synthesized polysaccharides (%) in the 1-5 h culture. Compositional percentages are calculated by deconvoluting 1D 13 C CP spectra. ( E ) 2D 13 C- 13 C CORD spectra of the 5 h culture, with blue circles indicating the absence of β-1,3-glucan signals. ( F ) Cytochemical labeling of gold-specific staining for β-1,3-glucan (with Fc-Dectin-1, top), chitin (with WGA-lectin, middle), and anti-chitosan rabbit antiserum (bottom). Scale bars: 500 nm. Electron-dense gold particles (black dots) mark probe binding. The newly formed thin cell wall of germ tube (orange arrowheads) is labeled by WGA and anti-chitosan. Dectin labeling is only positive in the cell wall of the original resting conidium cell wall. Magenta arrows indicate the location of outer wall changes, and yellow arrows highlight the extending inner layer contributing to germ tube formation.

Article Snippet: Grids were washed with endotoxin-free water and incubated for 1 h with 100 μg mL -1 recombinant human Fc-Dectin-1 (Fc-hDectin-1a, InvivoGen).

Techniques: Labeling, Synthesized, Staining, Binding Assay

Journal: bioRxiv

Article Title: Synergistic activation of TLR2 and Dectin-2 by mannose-capped lipoarabinomannan reprograms macrophage lipid metabolism in tuberculosis

doi: 10.64898/2026.02.18.706227

Figure Lengend Snippet: A. M. tuberculosis ManLAM (500 ng/ml), H 2 O 2 treated ManLAM (ML+H, 500 ng/ml), furfurman (FUR; commercial Dectin-2 agonist; 10 µg/ml) and vehicle control (C) were tested were tested for ability to induce NF-κB activation in HEK-Dectin-2 reporter cells. Data were generated and analyzed as in . B. Control and Dectin-2 knockdown (KD) iBMDM were treated 24h with 500ng/ml ManLAM preparations. Abbreviations are as in panel A. Imaging flow cytometry data were generated and expressed as in . C. Representative microscopy images (60x magnification) of vehicle-control (C) and treated iBMDM in the experiment shown in panel B. Panel description as in . D. iBMDM were treated with ManLAM (500 ng/ml) doses of furfurman (FUR; 2.5, 5 and 10 µg/ml). Imaging flow cytometry data were generated and expressed as in . E. Representative microscopy images (60x magnification) of vehicle-control (C) and treated iBMDM in the experiment shown in panel D. Panel description as in . *, p <0.05 by unpaired t test in all relevant panels.

Article Snippet: TLR2 ligand Pam3CSK4 and Dectin-2 ligand furfurman were purchased from Invivogen (San Diego, CA).

Techniques: Control, Activation Assay, Generated, Knockdown, Imaging, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: Synergistic activation of TLR2 and Dectin-2 by mannose-capped lipoarabinomannan reprograms macrophage lipid metabolism in tuberculosis

doi: 10.64898/2026.02.18.706227

Figure Lengend Snippet: A. iBMDM were treated with Pam3CSK4 (PAM; 500 ng/ml) and furfurman (FUR; 10 µg/ml), singly or in combination (PAM+FUR), and corresponding vehicle controls. Imaging flow cytometry data were generated and expressed as in . B. Representative microscopy images (60x magnification) of vehicle-control (C) and treated iBMDM in the experiment shown in panel A. Panel description as in . C. M. tuberculosis ManLAM, demannosylated ManLAM (αtManLAM), and deacylated ManLAM (dManLAM) (all H 2 O 2 pre-treated, 500 ng/ml) and corresponding vehicle controls were tested for ability to induce NF-κB activation in HEK-TLR2 and HEK-Dectin 2 reporter cells. Treatment was for 24h as in preceding figures. Data are presented as ratio of OD 650 values obtained with treated vs vehicle control cells. The horizontal dotted line marks the corresponding vehicle-control reading. D. ManLAM and derivatives, as in panel C, were used to treat iBMDM (all 500 ng/ml for 24h). Imaging flow cytometry data were generated and expressed as in . E. Representative microscopy images (60x magnification) of vehicle-control (C) and treated iBMDM in the experiment shown in panel D. Panel description as in . *, p <0.05 by unpaired t test in all relevant panels.

Article Snippet: TLR2 ligand Pam3CSK4 and Dectin-2 ligand furfurman were purchased from Invivogen (San Diego, CA).

Techniques: Imaging, Flow Cytometry, Generated, Microscopy, Control, Activation Assay

Journal: bioRxiv

Article Title: Synergistic activation of TLR2 and Dectin-2 by mannose-capped lipoarabinomannan reprograms macrophage lipid metabolism in tuberculosis

doi: 10.64898/2026.02.18.706227

Figure Lengend Snippet: A. iBMDM, control (C, non-targeting guide RNAs), TLR2 knock-down (KD), and Dectin-2 (Dec-2) KD iBMDM clones were left uninfected or infected with M. tuberculosis mc 2 6206 (MOI= 15) for 48h in triplicate wells, washed, and stained with Bodipy 493/503 and antibodies to the macrophage marker F4/80 for imaging flow cytometry. Data were generated and expressed as in . B. Representative microscopy images (60x magnification) of uninfected (U) and infected (Mtb) iBMDM in the experiment shown in panel A. Panel description as in . C, D. iBMDM were treated with ManLAM (500ng/ml) and vehicle-treated or treated with increasing doses of the diacylglycerol transferase inhibitor, A922500 (DGAT-i; 60, 90, 120ng/ml) and the acyl-coenzyme A:cholesterol acyltransferase inhibitor, CAS 615264-52-3 (ACAT-i; 5, 10, 15µg/ml). Imaging flow cytometry data for lipid droplet content were generated and expressed as in . *, p <0.05 by unpaired t test in all relevant panels. E. iBMDM were treated with ManLAM (500ng/ml) and vehicle-treated or treated with increasing doses of the PPARγ inhibitor GW9662 (0.5, 1, 2µM), mTORC1 inhibitor rapamycin (0.2, 0.4, 0.8nM), and the NF-κB inhibitor JSH-23 (3.5, 7, 14µM). Imaging flow cytometry data for lipid droplet content were generated and expressed as in . *, p <0.05 by unpaired t test in all relevant panels.

Article Snippet: TLR2 ligand Pam3CSK4 and Dectin-2 ligand furfurman were purchased from Invivogen (San Diego, CA).

Techniques: Control, Knockdown, Clone Assay, Infection, Staining, Marker, Imaging, Flow Cytometry, Generated, Microscopy

Journal: bioRxiv

Article Title: Synergistic activation of TLR2 and Dectin-2 by mannose-capped lipoarabinomannan reprograms macrophage lipid metabolism in tuberculosis

doi: 10.64898/2026.02.18.706227

Figure Lengend Snippet: Acyl groups and mannose caps within ManLAM engage TLR2 and Dectin-2, respectively, activating an mTORC1-PPARγ pathway that promotes lipid droplet accumulation and an NF-κB pathway that induces inflammatory cytokine production (e.g., TNF-α). Lipid metabolic reprogramming proceeds largely independently of inflammatory signaling.

Article Snippet: TLR2 ligand Pam3CSK4 and Dectin-2 ligand furfurman were purchased from Invivogen (San Diego, CA).

Techniques: