Western blot demonstrating the presence of the majority of Pmp18D in the insoluble material after sonication in PBS 48 h post-infection: a) anti-N-Pmp18D, b) anti-Mid-Pmp18D, c) anti-C-Pmp18D and d) anti-Omp-1 mAb 4/11.

<p>Western blot demonstrating the presence of the majority of Pmp18D in the insoluble material after sonication in PBS 48 h post-infection: a) anti-N-Pmp18D, b) anti-Mid-Pmp18D, c) anti-C-Pmp18D and d) anti-Omp-1 mAb 4/11.</p

Schematic representation of Pmp18D cleavage products as revealed following proteomic analysis of protein fragments.

<p>Schematic representation of Pmp18D cleavage products as revealed following proteomic analysis of protein fragments.</p

Western blot demonstrating an increase in Pmp18D expression and proteolytic cleavage over the 72 h <i>C. abortus</i> developmental cycle using: a) anti-N-Pmp18D, b) anti-Mid-Pmp18D, c) anti-C-Pmp18D pAbs and d) anti-Omp-1 mAb 4/11.

<p>Western blot demonstrating an increase in Pmp18D expression and proteolytic cleavage over the 72 h <i>C. abortus</i> developmental cycle using: a) anti-N-Pmp18D, b) anti-Mid-Pmp18D, c) anti-C-Pmp18D pAbs and d) anti-Omp-1 mAb 4/11.</p

image_2_Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.tif

<p>Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders’ innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1^−/− mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.</p

image_1_Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.tif

<p>Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders’ innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1^−/− mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.</p

Identification of YhaO and YhaJ as potential virulence determinants.

<p>(A) Screening of the <i>yhaOMKJ</i> locus for a role in virulence. SDS-PAGE profile of secreted proteins from TUV93-0, <i>yhaO</i>, <i>yhaM</i>, <i>yhaK</i> and <i>yhaJ</i> cultured in MEM-HEPES. Arrows indicate the location of the major LEE-encoded secreted effectors Tir, EspD and EspA as identified by mass-spectrometry. Samples were normalized according to cellular OD⁶⁰⁰ to normalize loading into each well. Immunoblot analysis of EspD levels from secreted (Sec) and whole cell lysate (WCL) fractions confirmed the SDS-PAGE results. Anti-GroEL was used to verify equal concentrations of WCL, which corresponded to OD⁶⁰⁰ normalized culture samples, loaded into each well (B) SDS-PAGE analysis highlighting complementation of the <i>ΔyhaO</i> and <i>ΔyhaJ</i> phenotypes by plasmids p<i>yhaO</i> and p<i>yhaJ</i>. SDS PAGE and immunoblot analysis of secreted protein profiles and EspD cytoplasmic expression confirmed the results. Protein secretion experiments were performed on multiple occasions.</p

YhaJ directly regulates <i>yhaO</i> expression in EHEC.

<p>(A) Purified YhaJ was tested for its ability to bind the <i>yhaO</i> promoter region (<i>pyhaO;</i> ~300 bp region upstream of the <i>yhaO</i> coding sequence) by EMSA. DIG-labeled <i>pyhaO</i> was incubated with increasing concentrations of YhaJ that corresponded to a shift in free-DNA indicating a YhaJ-DNA complex. Specificity of the binding reaction was tested by the addition of a 100-fold excess (+) of unlabeled <i>pyhaO</i> probe to the binding reaction to outcompete binding of the DIG-labeled probe to YhaJ. These reaction conditions were carried out using a fragment of the <i>kan</i> gene as a negative control. Additionally, the unlabeled <i>kan</i> probe in 100-fold excess was used as a non-specific competitor for YhaJ binding to the DIG-labeled <i>yhaO</i> probe (p<i>yhaO</i> vs <i>kan</i>). (B) Activity of the <i>yhaO</i> promoter in the <i>ΔyhaJ</i> mutant background. A plasmid containing a GFP-<i>yhaO</i> promoter fusion was transformed into TUV93-0 and <i>ΔyhaJ</i> to monitor transcription of <i>yhaO</i> in RFU during growth in MEM-HEPES. Data was calculated from three biological replicates and plotted at increasing OD⁶⁰⁰ values. * denotes P ≤ 0.05.</p

Schematic model of LEE regulation by the YhaO/YhaJ D-serine sensory system.

<p>Summary of small molecule signals that are encountered by EHEC in the intestinal (red) and extraintestinal (blue) environments [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005359#ppat.1005359.ref009" target="_blank">9</a>]. In the intestinal environment LEE expression is affected by signals such as fucose, ethanolamine and quorum sensing molecules (epinephrine, norepinephrine and AI-3). YhaJ constitutively regulates <i>yhaO</i> as well as stimulating the LEE (+) helping to promote A/E lesion formation and colonization of host tissue. In the extraintestinal environment D-serine can be encountered in high concentrations leading to repression (-) of the LEE by an unknown (?) direct mechanism [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005359#ppat.1005359.ref021" target="_blank">21</a>]. Expression of <i>yhaO</i> is also increased resulting in further uptake of D-serine and thus a greater transcriptional response to this signal (+++) promoting inhibition of colonization in unfavorable environments. The outer membrane (OM), peptidoglycan layer (PG) and inner membrane (IM) of EHEC are indicated.</p

YhaO and YhaJ are required for attaching and effacing lesion formation on host cells.

<p>(A) Wide-field fluorescence microscopy images of HeLa cells incubated with TUV93-0, <i>ΔyhaO</i>, <i>ΔyhaO</i> + p<i>yhaO</i>, <i>ΔyhaJ</i> and <i>ΔyhaJ</i> + p<i>yhaJ</i> in MEM-HEPES (LEE-inducing conditions). Host cells were stained with FITC-Phalloidin to fluorescently label actin green (488) and bacterial cells were either transformed with a plasmid constitutively expressing RFP (<i>ΔyhaO</i> and <i>ΔyhaJ</i>) or stained with Alexafluor 555 (p<i>yhaO</i> and p<i>yhaJ</i>) to label them red. Merged channels clearly show the areas of localized actin condensation beneath colonized bacterial cells, which corresponds to A/E lesion and pedestal formation as indicated by a white arrow. (B) Quantification of the average percentage of colonized host cells in the <i>ΔyhaO</i> and <i>ΔyhaJ</i> mutants and corresponding complementation backgrounds relative to TUV93-0. (C) Quantification of the average percentage of attached bacteria forming A/E lesions on bound host cells. Data was calculated from three biological replicates with at least twenty-five random fields of view taken per replicate. ***, ** and * denote P ≤ 0.001, P ≤ 0.01 and P ≤ 0.05 respectively.</p

Genomic and phylogenomic context of the <i>yhaOMKJ</i> locus.

<p>(A) Genomic context of the D-serine tolerance locus (blue) in three distinct <i>E</i>. <i>coli</i> isolates–CFT073 (UPEC), EDL933 (EHEC) and MG1655 (K-12). The system encodes DsdC (a LysR type transcriptional regulator), DsdX (a D-serine outer membrane transporter) and DsdA (a D-serine deaminase). In EDL933 the D-serine tolerance locus is truncated and replaced with the sucrose utilization locus (<i>cscRAKB</i> highlighted in green). (B) Genomic context of the second putative D-serine sensory locus (red) in CFT073, EDL933 and MG1655. The system encodes YhaJ (a putative LysR type transcriptional regulator), YhaK (a redox-sensitive bicupin), YhaM (a putative deaminase) and YhaO (a putative inner membrane D-serine transporter). (B) The <i>yhaOMKJ</i> locus is highly conserved across the <i>E</i>. <i>coli</i> phylogeny. Circularized phylogenomic tree of 1591 <i>E</i>. <i>coli</i> and <i>Shigella</i> isolates overlaid with gene carriage for the <i>dsdCXA</i> locus and the <i>yhaOMKJ</i> locus. The <i>yhaOMKJ</i> genes are indicated by red blocks and the <i>dsdCXA</i> locus by blue blocks. Ordering of the genes is numbered and corresponds to the gene in the legend labeled *. Presence of a gene is determined by > 80% identity over > 80% of the coding sequence. Pseudogenes are indicated as yellow blocks. <i>E</i>. <i>coli</i> phylogroups are subdivided by color with the branch point labeled on the tree. Phylogroup A = Blue; Phylogroup B1 = Green; Phylogroup B2 = Red; Phylogroup C = Magenta; Phylogroup D = Purple; Phylogroup E = Cyan; Phylogroup F = Brown; <i>Shigella</i> = Gold. The position of prototypical strains is indicated on the outside of the figure.</p