Journal: Toxins
Article Title: ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo
doi: 10.3390/toxins13090645
Figure Lengend Snippet: Cyclic-di-GMP regulates ExlA secretion( A ). Intracellular c-di-GMP levels in IHMA::pSW196 (IHMA) and PAO1::pSW196 (PAO1). Both strains contained the chromosomal PcdrA-gfp (ASV) c fusion as fluorescent reporter of c-di-GMP levels. GFP fluorescence was recorded for 16 h and was normalized by OD 585 to assess bacterial growth. The results, in arbitrary units (A; U), represent the mean +/− SD of six replicates. ( B ). The areas under the curves (AUC) were deduced from data shown in ( A ). Bar: mean. The indicated p -value was calculated with the Student’s test. ( C , D ). Similar experiment using IHMA::pSW196 (IHMA), IHMA::pSW196- wspR* (c-di-GMP +) and IHMA::pSW196- PA2133 (c-di-GMP −). Gene expression was induced by arabinose 0.025%. Statistical differences between AUC were calculated with ANOVA ( p < 0.0001) followed by Dunnett’s test for comparison with IHMA. ( E ). ExlA contents of secretomes and bacteria for IHMA::pSW196 (WT), IHMA Δ exlBA , IHMA::pSW196- wspR* (c-di-GMP +) and IHMA::pSW196- PA2133 (c-di-GMP −). Secretomes were concentrated 100X and bacterial extracts 10X before Western blot analysis and incubation with ExlA antibodies (Cter and ΔCter). FliC and EFTu were used as loading controls for secretomes and bacterial extracts, respectively. ( F ). ExlA/control signal ratios in secretomes and bacterial extracts are shown for three independent Western blot experiments (color coded). ( G ). ExlA was quantified by ELISA in the secretomes of IHMAΔ erfA ::pSW196 (Δ erfA ), IHMAΔ erfA ::pSW196- wspR* (c-di-GMP +) and IHMAΔ erfA ::pSW196- PA2133 (c-di-GMP −). Three clones were assayed in triplicates. The dots represent the data for each clone with the mean (bar). Global statistical difference was established with ANOVA ( p = 0.0036) and p -values for individual comparisons with IHMAΔ erfA (Dunnett’s test) are shown. ( H ). The three IHMA derivatives described in ( C , D ) (WT, c-di-GMP + and c-di-GMP −) were analyzed in strains harboring lacZ integrated within exlA gene to measure the transcriptional activity of the exlBA promoter. The β-galactosidase activity was measured in triplicates when bacterial cultures reached OD 600 = 1 and expressed in Miller’s units (MU). Results are shown as mean +/− SD. No significant differences were established with ANOVA. ( I ). ExlA contents in bacterial extracts (concentrated 10X) from strains IHMA exlB mut::pSW196 (Δ exlB ), IHMA exlB mut::pSW196- wspR* (Δ exlB c-di-GMP +), IHMA exlBmut ::pSW196- PA2133 (Δ exlB c-di-GMP −) were determined by Western blot. EFTu was used as loading control.
Article Snippet: Membranes were incubated with the following antibodies: Cter and ΔCter ExlA antibodies, FliC [ ], EFTu (Hycult Biotech, HM6010), DsbA and Opr86 [ ].
Techniques: Fluorescence, Expressing, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Clone Assay, Activity Assay
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