BapA and BapC are secreted in a TTSS3-dependent manner.

<p>(A) Proteins in supernatant samples from mid-exponential growth phase cultures of the wild-type ([<i>bapA</i>TC], [<i>bapC</i>TC], [<i>bopE</i>TC] and [pBHR1]) or <i>ΔbsaS</i> (<i>ΔbsaS</i>[<i>bapA</i>TC], <i>ΔbsaS</i>[<i>bapC</i>TC], <i>ΔbsaS</i> [<i>bopE</i>TC] and <i>ΔbsaS</i>[pBHR1]) strains were precipitated using DOC/TCA and labeled with the FlAsH reagent. (B) Proteins in unlabeled samples were separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Protein bands at or near the expected size of TC-tagged BapA (120 kDa), BapC (23 kDa) and BopE (33 kDa) are indicated by the boxes. The positions of molecular mass markers are shown on the left.</p

Identification of TC-tagged BapA and BapC by FlAsH labelling of live <i>B</i>. <i>pseudomallei</i>.

<p>Proteins in whole cell lysates of FlAsH-labeled culture samples were separated by SDS-PAGE and visualized by (A) fluorescence or (B) Coomassie Blue staining. Protein bands at or near the expected size of TC-tagged BapA (120 kDa), BapC (23 kDa) and BopE (33 kDa) are indicated by the boxes. The positions of molecular mass markers are shown on the left. Vertical black lines on the gel image indicate that unrelated intervening lanes have been removed.</p

Competitive <i>in vivo</i> growth indices for each of the <i>ΔbapA</i>, <i>ΔbapB</i> and <i>ΔbapC</i> mutant strains and the <i>ΔbapB</i>[pBHR1] and <i>ΔbapB</i>[<i>bapB</i>] complemented strains compared to the wild-type strain.

<p>Each point on the graph indicates the competitive index (CI) measured in a single mouse. The horizontal marks show the average CI ± 1 SEM. *<i>P</i> < 0.05.</p

Transcription of <i>bopE</i> during the early (A) -, mid (B)—and late (C) -exponential growth phases.

<p>The level of <i>bopE</i> transcription in each sample was normalized to the expression of the housekeeping gene <i>rpoA</i>. The different strains analyzed were: <i>ΔbapA</i>, the <i>ΔbapA</i> strain; <i>ΔbapB</i>, the <i>ΔbapB</i> strain; WT, the wild-type strain. Data are expressed as mean ± SEM. Error bars represent the SEM from three technical replicates of biological duplicates. *<i>P</i> < 0.05, **<i>P</i> < 0.001, ***<i>P</i> < 0.0001.</p

BopE expression and secretion during early- (A, B), mid- (C, D) and late-exponential (E, F) growth phases.

<p>Supernatant or total culture samples were separated by SDS-PAGE and transferred to a membrane prior to western immunoblotting using anti-BopE antiserum. The relative expression levels, determined by densitometric analysis of the western blots and normalized against the wild-type expression, are presented as the mean ± SEM of three biological replicates. *<i>P</i> < 0.05, ***<i>P</i> < 0.0001. A single representative western blot is shown above the quantified averaged data for comparison. A vertical black line on the western blot indicates unrelated intervening lanes have been removed. The samples were derived from the <i>ΔbapA</i>, <i>ΔbapB</i>, <i>ΔbapC</i>, <i>ΔbopE</i> or the the wild-type (WT) strain.</p