Journal: mBio
Article Title: The Bordetella effector protein BteA induces host cell death by disruption of calcium homeostasis
doi: 10.1128/mbio.01925-24
Figure Lengend Snippet: BteA-induced cell death is characterized by fragmentation of the endoplasmatic reticulum and mitochondrial networks. ( A ) Time lapse analysis of morphological changes and plasma membrane permeabilization in Hela cells. Hela cells were infected with B. bronchiseptica wild-type ( Bb WT) and Bb Δ bteA mutant ( Bb Δ bteA ), expressing the fluorescent protein mNeonGreen, at MOI of 10:1 in the presence of propidium iodide (5 µg/mL). A sequence of time lapse images is shown. Bright field, gray; bacteria, cyan; propidium iodide, magenta. Scale bar, 20 µm. Data are representative of three independent experiments. ( B and C ) Comparison of B. bronchiseptica and B. pertussis cytotoxicity against HeLa cells. HeLa cells were infected with B. bronchiseptica ( B ) or B. pertussis ( C ) wild-type strains and their mutant derivatives at the indicated MOI. Plasma membrane permeabilization was determined using the fluorescent DNA binding dye CellTox Green. For B. bronchiseptica , infections were conducted both in the presence (gly+) and absence of glycine (5 mM). Asterisks indicate a statistically significant difference ( P < 0.05, unpaired two-tailed t -test) between Bb WT and Bb WT gly+ infection samples at 4 h. Data represent the mean ± SEM of a representative experiment out of 2 performed in technical triplicate. ( D ) Visualization of cellular structures. Hela cells were transfected to express fluorescent proteins tagged with localization signals for endoplasmatic reticulum (ER) and mitochondria (mito). One hour after infection with Bb WT and Bb Δ bteA mutant at MOI 50:1 , cells were fixed and analyzed by fluorescence imaging. ER, yellow; mitrochondria, magenta. Scale bar, 20 µm. The shown micrographs are representative of two independent experiments from which the organelle morphology was scored. Analysis was performed on at least 100 cells per experiment and condition and is plotted as morphology % ± SEM. Asterisks indicate statistically significant differences ( P < 0.05, unpaired two-tailed t -test) between the % of fragmented ER or mito compared with uninfected cells.
Article Snippet: The GroES promotor (391 nt, NC_002927.3, 1 041 354–1 041 744) was amplified from chromosomal DNA of B. bronchiseptica RB50, whereas the mNeonGreen coding sequence was amplified from 4xmts-mNeonGreen vector (Addgene item # 98876) using Herculase II Phusion DNA polymerase (Agilent, USA).
Techniques: Clinical Proteomics, Membrane, Infection, Mutagenesis, Expressing, Sequencing, Bacteria, Comparison, Binding Assay, CellTox Assay, Two Tailed Test, Transfection, Fluorescence, Imaging
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