Journal: bioRxiv

Article Title: To Kill or not to Kill: A Conserved trans -intoxication protection factor Blocks X-T4SS-Mediated Fratricide through Interaction with VirB5

doi: 10.64898/2026.05.09.723464

Figure Lengend Snippet: (A) Colony competition between S. maltophilia wild-type or Δ vir D4 attacker strains and X. citri Δ8Δ2609Δ xac 2606Δ xac2611 prey cells expressing constitutive GFP and carrying either an empty pBRA vector, pBRA-Smlt2994, pBRA-Smlt2995, pBRA-Smlt2996, the pBRA plasmid encoding the entire operon smlt2994–smlt2995–smlt2996 [pBRA-Smlt299(4-5-6)], or pBRA-XAC2611. Mixed cultures were spotted on AB agarose medium, incubated for 7 days at room temperature, and fluorescence images were acquired using a Bio-Rad ChemiDoc MP imaging system. (B) Time-lapse fluorescence microscopy of X. citri versus S. maltophilia competition. X. citri Δ8Δ2609Δ xac 2606Δ xac2611 GFP (green cells) carrying an empty pBRA vector, pBRA-Smlt2994, pBRA-Smlt2995, or pBRA-Smlt2996 are shown. Two frames from each movie are displayed, corresponding to the start (T = 00:00 h) and final (T = 08:00 h) time points. Additional examples of competition are shown in Supplementary Movies X. Strains and plasmid constructs used in each assay are indicated in the figure labels. Scale bars, 5 µm. (C) Quantitative analysis of the bacterial competition observed in Supplementary Movies S14–S17 . Bars represent the mean percentage of visually identified X. citri GFP-positive cells and S. maltophilia cells at the onset of the experiment (T = 00:00 h), after 4 h (T = 04:00 h), and after 8 h (T = 08:00 h), scored across four to five representative microscopy fields (18 µm × 18 µm). Symbols denote values for each field, bars indicate the mean, and error bars represent the standard deviation. Across all conditions, a total of 1,128, 1,717, and 2,220 cells ( X. citri plus S. maltophilia ) were scored at T = 0, 4, and 8 h, respectively: for X. citri pBRA-Smlt2994 versus S. maltophilia , 313, 453, and 496 cells; for X. citri pBRA-empty versus S. maltophilia , 331, 570, and 721 cells; for X. citri pBRA-Smlt2995 versus S. maltophilia , 230, 265, and 451 cells; and for X. citri pBRA-Smlt2996 versus S. maltophilia , 254, 429, and 552 cells at T = 0, 4, and 8 h, respectively. At T = 00:00 h, the proportion of X. citri cells did not differ significantly among strains (one-way ANOVA, p = 0.51). By T = 04:00 h and T = 08:00 h, two clearly distinct groups emerged: a protected group, composed of X. citri Δ8Δ2609Δ xac 2606Δ xac2611 GFP cells expressing pBRA-Smlt2994 or pBRA-Smlt2996, which maintained ∼55% and ∼38–47% of the mixed population, respectively; and a susceptible group, composed of cells carrying the empty pBRA vector or pBRA-Smlt2995, which declined sharply to ∼14–23% and ∼4–8%, respectively (one-way ANOVA, p = 5.1 × 10[[ and p = 7.0 × 10[[; Tukey’s test, p = 0.0021–0.021 and p = 2.5 × 10[[–0.0017, at T = 04:00 h and T = 08:00 h, respectively). No significant differences were detected between strains within either group at any time point, indicating that Smlt2994 and Smlt2996 confer equivalent and robust protection, while Smlt2995 fails to protect X. citri from S. maltophilia X-T4SS-mediated killing. Scale bars (5 µm)

Article Snippet: Then, the membrane was washed with TBS, and Images were collected using the ChemiDoc System MP (BioRad).

Techniques: Expressing, Plasmid Preparation, Incubation, Fluorescence, Imaging, Microscopy, Construct, Standard Deviation

Journal: Nature Communications

Article Title: MAIT cells protect against pulmonary Legionella longbeachae infection

doi: 10.1038/s41467-018-05202-8

Figure Lengend Snippet: Human MAIT cells are activated by Legionella infection via MR1 in vitro. a Jurkat.MAIT and C1R.MR1 cells were co-incubated for 16 h with lysates of L. pneumophila (L. pn.) or L. longbeachae or 5-OP-RU, acetyl-6-formylpterin (Ac-6-FP) or PBS. Activation, detected by staining with anti-CD69, is enhanced by bacterial lysate or by the activating ligand 5-OP-RU, but not by acetyl-6-FP. Activation was blocked by anti-MR1 antibody (26.5) but not by isotype control (W6/32) 2 h prior to co-incubation. Experiment performed in triplicate wells on two separate occasions with similar results. Data show mean fluorescence intensity, MFI (±SEM). Statistical tests: one-way ANOVA and post hoc Dunnett’s comparing all columns with the first column (black). Unpaired t -test (blue), with *** P < 0.001; **** P < 0.0001. b , c THP1 cells (WT) or THP1 cells overexpressing MR1 (THP1.MR1+, purple) or deficient in expression of MR1 (THP1.MR1−, blue) were infected for 27 h with live or heat-killed (HK) L. longbeachae (MOI: 100) or 10 nM 5-OP-RU, then co-cultured for 16 h with sorted CD3 + Vα7.2 + CD161 + human peripheral blood MAIT cells, or MAIT-depleted conventional T cells. MR1-5-OP-RU-tetramer+ MAIT cell activation was measured by intracellular cytokine staining for b TNF or c IFN-γ. b , c Percentage cytokine-positive cells as mean (±SEM) data from three independent donors performed on two separate occasions are shown. Statistical tests: unpaired t- tests with Bonferroni corrections, each comparing against MOI 0 for the specific cell line. Statistics with * P < 0.05; ** P < 0.01. d Immunofluorescence micrographs showing CD3+TCRVα7.2+ MAIT cell (white arrow) within healthy human lung tissue (top panel) and 24 h post infection (bottom panels) ex vivo with L. longbeachae . Yellow arrow: intracellular L. longbechae bacilli. Red, TCRVα7.2; green, CD3; magenta, polyclonal rabbit anti -L. longbeachae ; blue, nuclei (Hoechst)

Article Snippet: Following rehydration, the unfixed cryopreserved 8 μm sections of human lung were blocked with serum-free block and stained with purified anti-Vα7.2 TCR mAb clone 3C10 (Biolegend), rat anti-human CD3 mAb (BioRad) and polyclonal rabbit anti- Legionella antibody.

Techniques: Infection, In Vitro, Incubation, Activation Assay, Staining, Control, Fluorescence, Expressing, Cell Culture, Immunofluorescence, Ex Vivo