List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.

<p>List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.</p

Transcriptome changes elicited by glucose in GBS.

<p>Comparison of gene expression changes (log2) between mid-log cultures of 2603 V/R wild-type strain and <i>CovRS</i> mutant following challenge with or without glucose. Data for the wild type and mutant strains are shown on the <i>x</i> axis and <i>y</i> axis, respectively.</p

Real-time RT-PCR evaluation of <i>bibA</i> expression in 2603V/R and Δc<i>ovRS</i> strains grown in medium containing 55 mM glucose or in sugar-free medium.

<p>Transcript levels were normalized to the expression level of <i>gyrA</i>. Syber green runs were performed with cDNAs from the same reverse transcription reaction from 1 µg of total RNA. The ΔΔCT method was applied as a comparative method of quantification, using strains grown in sugar free medium as reference. The data are representative of 2 independent experiments, each in triplicate. Error bars, SD.</p

Graphical representation summarizing adaptive regulation of GBS in high glucose conditions.

<p>Genes of interest are color-grouped according to main functional categories. Arrows indicate up- or down-regulation relative to time of 30′ in high glucose <i>vs.</i> no glucose.</p

Differential regulation of gene expression in GBS strain 2603 V/R versus the isogenic Δ<i>CovRS</i> mutant strain after incubation in medium with 55 mM glucose versus a sugars-free complex medium.

<p>White bars indicate the number of glucose-regulated genes in the wild-type strain; black bars indicate the number of genes that are glucose- dependent and CovRS-dependent; grey bars indicate the number of genes that are glucose-dependent and CovRS-independent.</p

CovR binds to <i>bibA</i> promoter <i>in vivo</i>. (A)

<p>Quantification by qRT-PCR of <i>bibA</i> promoter immunoprecipitated with CovR antiserum in 2603 V/R wild type strain grown in medium devoid of glucose or in the presence of 55mM glucose. <i>cfb</i> promoter and <i>cylX</i> promoter were used as a positive control while <i>sag0017</i> promoter was used as a negative control. The level of PCR products of eluate from the isogenic Δc<i>ovRS</i> deletion mutant grown with or without glucose was negligible. The data are representative of 3 independent experiments, each in triplicate. Error bars, SD. (<b>B)</b> Competitive EMSA experiment. Labelled <i>PbibA</i> fragment (3.3 nM) was incubated without <i>(lane1)</i> or with CovR (2 µM) <i>(lane2</i>–<i>6)</i>, in the presence of different amounts of unlabelled <i>PbibA (lane 3</i>–<i>4)</i>, as a specific competitor, and <i>Psag0017 (lane5</i>–<i>6)</i>, as a non-specific competitor. The labelled DNA was detected by chemioluminescence. <b>(C)</b> CovR phosphorylation increases its affinity for <i>bibA</i> promoter. Electrophoretic mobility shift assay using recombinant CovR (left) and chemically phosphorylated recombinant CovR (right). Labelled <i>PbibA</i> DNA fragment (3.3 nM) was incubated without or with the indicated amounts of CovR. The labelled DNA was detected by chemioluminescence.</p

SslE Elicits Functional Antibodies That Impair <i>In Vitro</i> Mucinase Activity and <i>In Vivo</i> Colonization by Both Intestinal and Extraintestinal <i>Escherichia coli</i> Strains

<div><p>SslE, the <u>S</u>ecreted and <u>s</u>urface-associated <u>l</u>ipoprotein from <i><u>E</u>scherichia coli</i>, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of <i>in vitro</i> bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired <i>E. coli</i> mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslE_IHE3034), are able to inhibit translocation of <i>E. coli</i> strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslE_IHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in <i>E. coli</i> colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic <i>E. coli</i> species.</p></div

The <i>sslE</i> promoter is functional in an intestinal model of colonization.

<p>(A) 2D <i>in vivo</i> imaging at 24 hours of mice intragastrically infected with GL53-P<i>neg</i>-<i>luxCDABE</i> (promoterless control vector), with the bioluminescent derivative GL53-P<i>sslE</i>-<i>luxCDABE</i> and with the GL53-P<i>em7-luxCDABE</i> (positive control). (B) 3D image reconstruction showing <i>ssIE</i>-promoter driven luciferase expression in <i>E. coli</i> localized in the intestinal tract. (C) RT-PCR of RNA purified from: <i>in vitro</i> lab-grown GL53 bacteria (lane 2, positive control); caecum tract of uninfected mice (lane 3, negative control); GL53 bacteria recovered from infected mice (lane 4); GL53 bacteria recovered from infected mice without the RT step (lane 5). 1 Kb Plus DNA Ladder (Life Technologies) is shown in lane 1.</p

Cross-inhibition of <i>E. coli</i> translocation through a mucin-gel matrix by anti-SslE_IHE3034 (belonging to variant I) antibodies.

<p>EPEC IC50 (A), EAHEC LB226692 (B), ETEC GL53 (C), SEPEC IN1S (D) strains carrying variant II were loaded on top of the gel-mucin matrix column and bacterial translocation with or without anti-SslE_IHE3034 antibodies was assessed in three sequentially collected fractions. For each strain, translocation was reported as the percentage of CFU recovered with respect to the initial inoculum.</p

SslE_IHE3034 induces cross-protection in intestinal colonization, UTI and sepsis models.

<p>(A) Thirty CD1 mice were intranasally immunized with 30 µg of SslE_IHE3034 at days 1, 21 and 35. Saline was used in the negative control groups. Challenge was done by oral gavage with 5×10⁷ CFU of strain GL53 at day 49. Serial dilutions of the homogenized intestinal caecum tract were plated and the CFU number was enumerated. Statistical significance of protection was obtained using the Mann Whitney test. (B) SslE_IHE3034 prevents the spread of the UPEC strain 536 into the kidneys and spleen in an ascending model of urinary tract infection. Thirty mice were immunized intranasally with 10 µg cholera toxin (CT) alone or with 100 µg of SslE_IHE3034 at a 10∶1 ratio of antigen:CT (day 1). After two boosts of 25 µg antigen (10∶1 ratio of antigen to CT) or CT alone (day 7 and 14), mice were transurethrally challenged with 10⁸ CFU of strain 536 at day 21. After 48 h, bladder, kidneys and spleen were harvested and homogenized. Bacteria in urine and in the tissue homogenates were enumerated by plating serial dilutions. Symbols represent CFU/g tissue or CFU/ml urine of individual mice, and bars indicate median values. P values were determined using the nonparametric Mann-Whitney significance test. (C) SslE_IHE3034 protects against the SEPEC strain IN1S in a sepsis mouse model. CD1 out-bred mice were immunized by subcutaneous injections at day 1, 21, and 35 with 20 µg of recombinant SslE_IHE3034 formulated with alum or alum alone. Immunized animals were challenged at day 49 with a sublethal dose of heterologous strain IN1S and survival was monitored for up to 4 days. The results are indicated as percentage of survival out of a total number of 40 mice. P values were determined using the nonparametric Mann-Whitney significance test.</p