Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic <i>Escherichia coli</i> (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp) - Fig 1

<p>Graphical map of the plasmids pCss-E1189 (1A; Accession number LN908839), and pCss-E1373 (1B; Accession number LN908840). Virulence genes (in yellow), plasmid-specific genes for replication (in green) and for stability (in red), and the global regulator gene <i>rns</i> (in blue) are indicated. The plasmid-specific genes for stability found in pCss-E1189 were not found in plasmid pCss-E1373.</p

CS6+STh expressing ETEC isolates used in this study^*.

<p>CS6+STh expressing ETEC isolates used in this study^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152899#t002fn001" target="_blank">*</a>}.</p

CS6 phenotypic expression of CS6+STh ETEC isolates.

<p>CS6 phenotypic expression of CS6+STh ETEC isolates.</p

qRT-PCR analyses of differentially expressed genes of the S.

<div><p><b>Typhi Δ<i>yehUT</i> mutant</b>. </p> <p>A The relative expression levels of the 15 genes found to be differentially expressed on microarray analysis of the S. Typhi Δ<i>yehUT</i> mutant when compared to <i>S</i>. Typhi in low salt LB, at mid-log phase, at 37°C (Table S6 in File S1). The 15 genes examined are listed on the <i>y</i>-axis. Grey boxes < 1.0 indicates down regulated genes and grey boxes >1.0 indicates up regulated genes relative to <i>S</i>. Typhi. B qRT-PCR analysis showing relative expression levels of genes after complementation of the Δ<i>yehUT</i> mutant compared to <i>S</i>. Typhi (Table S6 in File S1).</p></div

A The <i>yehUT</i> operon with surrounding genes.

<p>The operon is 2349bp in length and located between 782210 to 784611 bp in <i>S</i>. Typhi Ty2 (Accession number AE014613) and between c2251460 to c253861 bp in <i>S</i>. Typhimurium SL1344 (Accession number FQ312003). The surrounding genes include <i>yehV</i>, <i>t0694</i>, <i>yehW</i>, <i>yehX</i> (upstream of <i>yehU</i>) and <i>yehS, t0699</i> and <i>yehR</i> (downstream from <i>yehT</i>). <b>B The domain organisation of the YehU and YehT proteins</b>. YehU protein comprises of five transmembrane (TM) alpha helical segments predicted using TMHMM webserver [17]. The intracellular signalling component consists of a GAF domain, a DHp (dimerization/histidine-containing phosphotransfer) domain that is connected to an ATP (adenosine triphosphate)-dependent Kinase domain. YehT protein consists of a Receiver domain and a LyTR-homologous DNA binding domain [16]. The phosphorylation sites are indicated (H, Histidine; D, Aspartate). The SNP positions identified in <i>yehU</i> and <i>yehT</i> genes are indicated below the proteins by grey boxes [10]. </p

SDS-PAGE and western blot analysis of epitope-tagged YehT protein from <i>S</i>.

<div><p><b>Typhi BRD948</b>. </p> <p>Proteins from whole cell bacterial lysates were transferred after electrophoretic separation in a 12% SDS-PAGE gel (Invitrogen) onto a nitrocelluose membrane (Invitrogen) and probed with anti-FLAG M2 monoclonal antibodies (Sigma). Lanes: (M) protein molecular weight markers (SeeBlue Plus2 Pre-stained standard; Invitrogen); (-) proteins extracted from <i>S</i>. Typhi BRD948 (negative control); (+) proteins extracted from <i>S</i>. Typhimurium SL1344 Δ<i>fliA</i> (positive control, 27.4kDa); FLAG proteins extracted from <i>S</i>. Typhi Δ<i>yehT</i>-tagged (27.4kDa). Bacteria was grown in low salt LB; normal salt LB; SPI-2 inducing; SPI-2 + 0.5% glucose; SPI-2 + 0.25% acetate overnight, shaking at 37°C.</p></div

SDS-PAGE and western blot analysis of epitope-tagged YehT protein from <i>S</i>.

<div><p><b>Typhimurium ST4/74</b>. </p> <p>Proteins from whole cell bacterial lysates were transferred after electrophoretic separation in a 12% SDS-PAGE gel (Invitrogen) onto a nitrocelluose membrane (Invitrogen) and probed with anti-FLAG M2 monoclonal antibodies (Sigma). Lanes: (M) protein molecular weight markers (SeeBlue Plus2 Pre-stained standard; Invitrogen); (-) proteins extracted from <i>S</i>. Typhimurium ST4/74 (negative control); (+) proteins extracted from <i>S</i>. Typhimurium SL1344 Δ<i>fliA</i> (positive control; 27.4kDa); FLAG proteins extracted from <i>S</i>. Typhimurium Δ<i>yehT</i>-tagged (27.4kDa). Bacteria was grown in low salt LB; normal salt LB; SPI-2 inducing; SPI-2 + 0.5% glucose; SPI-2 + 0.25% acetate overnight, shaking at 37°C.</p></div

Confocal fluorescence microscopy image of S. Typhimurium SL1344 and S. Weltevreden in epithelial cells.

<p>Image shows S. Typhimurium SL1344 (a; two hours and b; six hours) and S. Weltevreden (c; two hours and d; six hours) interacting with Hep-2 cells at two and six hours post exposure. Cell nuclei are stained with DAPI (blue), common surface antigens (CSA) on extracellular Salmonella bacteria are stained in red and the Salmonella (pSsaG) where GFP expressing and are visible in green (GFP).</p

Source and location of <i>Salmonella</i> Weltevreden isolates contributing to this study.

<p>Source and location of <i>Salmonella</i> Weltevreden isolates contributing to this study.</p

Viable Salmonella recovered in gentamicin killing assay with epithelial cells.

<p>a) Plots showing viable Salmonella (CFU/mL) recovered two (black) and six hours (grey) post infection after an invasion assay with Hep-2 cells infected with S. Typhimurium SL1344, S. Weltevreden SW C2346, SW 10259 SW98_11262 and SW99_3134 (MOI, 50); error bars indicate standard deviation. b) Plot of six hour time point only. Two-way ANOVA multiple comparisons were performed, significance is highlight by the symbols (* p<0.05, **** p<0.0001).</p