Estimated mean faecal bacterial shedding following oral challenge of immunized calves with <i>E</i>. <i>coli</i> O157:H7.

<p>Graphs represent the triplicate fecal bacterial count data from colonized calf samples only, defined by having at least one non-zero observation from the direct plating method, or one positive observation from the broth enrichment method. (A) Estimates of linear predictor (logarithm of CFU/g feces) for each immunized group at each day post-challenge. (B) Observed mean counts (open circles) (logarithm of CFU/g feces), the estimated mean counts (plus symbols joined by line) and 95% confidence intervals envelop (shaded region) for each immunized group at each day post-challenge. All observed means with zero CFU/g feces count are presented as -15 on the logarithmic scale. Broken horizontal dash line shows the desirable CFU/g feces threshold of 10⁴ (or 9.21 on log scale) CFU/g, below which is predicted to reduce the basic reproductive ratio below 1.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128391#pone.0128391.ref037" target="_blank">37</a>].</p

Summary of immunization protocols.

<p>Summary of immunization protocols.</p

Estimated mean proportions of colonized calves following oral challenge of immunized calves with <i>E</i>. <i>coli</i> O157:H7.

<p>(A) Estimates of linear predictor (logit of proportion of colonized calves) for each immunized group at each day post-challenge. (B) Observed mean proportions (open circles), the estimated mean proportions (plus symbols joined by line) and 95% confidence intervals envelop (shaded region) of colonized calves for each immunized group at each day post-challenge.</p

Cross-reactivity of vaccine-induced antibodies with secreted and whole cell antigen preparations from non-O157 EHEC serotypes.

<p>Representative western blot of (A) secreted protein preparations and (B) whole cell preparations from EHEC serotypes O111, O103:H2, O145, O26:H11, O26:H- and O26 with serum collected from a calf collected 2 days prior to first immunization (Pre-immunization) and 6 days after the final immunization (Post-immunization) with recombinant EspA, intimin and Tir. Bound IgG1 was visualized by chemiluminescence. The putative identities of EspA, Tir and intimin-reactive bands are indicated in brackets.</p

Serum antibody titres of different immunization groups.

<p>Mean titres of (A) IgA, (B) IgG1 and (C) IgG2 of EspA-, Intimin-, Tir- and H7 flagellin (H7)-specific antibodies within the serum of calves 2 days prior to the first immunization (Pre) and 6 days after the final immunization (Post) with either EspA, intimin and Tir (<i>E-I-T</i>), EspA and intimin (<i>E-I</i>), EspA, intimin and H7 flagellin (<i>E-I-H7</i>), or adjuvant alone (Control). Each bar represents the mean (on log₁₀ scale) and corresponding error bar shows the standard error of mean. Overall statistical significance of immunization group for each antibody titre post-immunization was assessed using a linear mixed model and indicated by a <i>p</i>-value below each graph. The mean difference in titres between pre- and post-immunization for each immunization group was investigated using a linear mixed model, and statistically significant (adjusted for false discovery rate, <i>p</i>_<i>f</i> < 0.05) changes are indicated by an asterisk (*).</p

Correction: Elucidation of the RamA Regulon in <i>Klebsiella pneumoniae</i> Reveals a Role in LPS Regulation

Correction: Elucidation of the RamA Regulon in <i>Klebsiella pneumoniae</i> Reveals a Role in LPS Regulatio

Venn diagram representing the RNA sequencing results.

<p>Ecl8Δ<i>ramA</i> or Ecl8Δ<i>ramR</i> were used as calibrators in the pairwise comparisons. The arrows ⬇ indicates a lower than 0.5 fold decrease in transcription compared to calibrator; ⬆ indicates a higher than 2 fold transcription compared to calibrator. The numbers beneath A, B and C indicate the number of transcripts showing higher or lower transcription (based on statistical cut-off) compared to calibrator. The genes under the different categories A, B and C represent pairwise comparisons between Ecl8/Ecl8Δ<i>ramA</i>, Ecl8Δ<i>ramR</i>/Ecl8Δ<i>ramA</i> and Ecl8/Ecl8Δ<i>ramR</i> comparison respectively; the genes in Area AB were found to be differentially transcribed in both the Ecl8/Ecl8Δ<i>ramA</i> and the Ecl8Δ<i>ramR</i>/Ecl8Δ<i>ramA</i> comparisons; the genes in Area AC were found to be differentially transcribed in both the Ecl8/Ecl8Δ<i>ramA</i> and the Ecl8/Ecl8Δ<i>ramR</i> comparisons; the genes found in the area BC were found to be differentially transcribed in both the Ecl8/Ecl8Δ<i>ramR</i> and Ecl8Δ<i>ramR</i>/Ecl8Δ<i>ramA</i> comparisons. The <i>romA</i> gene in Area ABC was found to be differentially transcribed in all the three comparisons.</p

A: Northern blot analysis of sRamA5.

<p>15 μg of total RNA extracted from Ecl8/pGEMTpI+<i>romA</i> were loaded into wells. The blots were either incubated with the ³²P-end labelled sRamA5 specific DNA probe or <i>romA</i> specific DNA probe. The bands pointed as 1 and 2 are primary transcripts for both the RNA codes for sRamA5 and <i>romA</i>. The band referred to as sRamA5 is specifically detected by the sRamA5 DNA probe, sized at around 60 nucleotides; the band referred to as <i>romA</i> was specifically detected by the <i>romA</i> DNA probe. <b>B:</b> EMSA of RamR-sRamA5 or RamR-PI interaction in the presence of sRamA5. (i). RamR-sRamA5 interaction. The concentrations of sRamA5 and RamR were 40 nM and 1 μM respectively. (ii). RamR-pI interaction in the absence/presence of sRamA5. Radioactive labeled pI was 2 nM from lane 1 to 4. RamR’s concentrations from lane 1 to 4 were: 0, 2, 0, 2 μM. Cold sRamA5’s concentrations from lane 1 to 4 were: 0, 0, 1, 1 μM. F_R = free RNA, C_R = RNA-protein complex, F_D = free DNA, C = RNA-DNA-protein complex. <b>C:</b> qPCR for the level of <i>romA</i> and sRamA5’s transcriptions in Ecl8Δ<i>ramR</i>. qPCR using LNA probe for determining the levels of sRamA5 transcription in Ecl8 and Ecl8Δ<i>ramR</i>. Despite sharing the same TSS, the transcript levels of sRamA5 are not linked to <i>romA</i> levels, thereby reducing the likelihood of sRamA5 being a 5’ untranslated region of <i>romA</i>. The log₂ fold changes in Ecl8Δ<i>ramR</i> displayed in the bar chart are relative to their transcript levels in Ecl8. One-way ANOVA analyses (P<0.001) were performed to demonstrate statistical significance. <b>D:</b> qPCR assay for the level of <i>ramR</i>, <i>romA</i> and sRamA5 in Ecl8Δ<i>ramR</i> pACYC<i>ramR</i> and Ecl8Δ<i>ramR</i> pACYC177. The log₂ fold changes in the two strains displayed in the bar chart are relative to their transcript levels in Ecl8Δ<i>ramR</i>. sRamA5 levels are elevated in the presence of <i>ramR</i>, implying that RamR could stabilise the sRamA5 transcript. One-way ANOVA analyses (P<0.001) were performed to demonstrate statistical significance.</p

A: Attachment of <i>K. pneumoniae</i> Ecl8, Ecl8Δ<i>ramA</i>, Ecl8Δ<i>ramR</i> or Ecl8∆ramRA to murine macrophage RAW 264.7 cell line.

<p>One-way ANOVA analyses were performed to demonstrate statistical significance. <b>B:</b> Microscopy to assess attachment to RAW 264.7 cell line. (i) Infection of the RAW264.7 cell line was carried out with <i>K. pneumoniae</i> Ecl8 (WT), Ecl8Δ<i>ramA</i>, Ecl8∆<i>ramR</i> or Ecl8Δ<i>ramRA</i> transformed with plasmid pRSMgfp. MOI was 1:100 and infections were carried out for 2 hrs. The actin cytoskeleton was stained with Acti stain 555 phalloidin (red) and host cell nuclei were stained with DAPI (blue). Images are representative of 80 fields. (ii) Graph representating mean values are derived from 3 independent experiments. One-way ANOVA analyses (P<0.001) were performed to demonstrate statistical significance. <b>C:</b> Internalisation of <i>K. pneumoniae</i> Ecl8, Ecl8Δ<i>ramA</i>, Ecl8Δ<i>ramR</i> or Ecl8∆<i>ramRA</i> by RAW 264.7 cells. Bacterial internalisation was assessed by the gentamicin protection assay. One-way ANOVA analyses were performed to demonstrate statistical significance. <b>D:</b> Enumeration of the extracellular non-phagocytosed <i>K. pneumoniae</i> Ecl8, Ecl8Δ<i>ramA</i>, Ecl8Δ<i>ramR</i> or Ecl8∆<i>ramRA</i>. One-way ANOVA analyses were performed to demonstrate statistical significance.</p

Strains used in this study.

<p>Strains used in this study.</p