Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors

Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Induction of filopodia by EPEC effector mutants with integrated <i>map</i>.

<p>Immunofluorescence microscopy of Swiss 3T3 cells infected for 10 min with EPEC2, EPEC1 and EPEC0 and isogenic strains with <i>map</i> integrated in the chromosome. EPEC was detected with rabbit polyclonal anti-O127 (red) and actin was stained with Oregon-green phalloidin (green). Filopodia spikes are labelled with arrowheads. Actin accumulation beneath bacteria is indicated with arrows. Scale bar 2 μm.</p

Deletion of effector genes in EPEC E2348/69.

<p><b>A</b>. Representation of the LEE pathogenicity island. Effector genes are labeled in red. The order of deletion is numbered: deletion 1, 2, 3, 4 are for <i>map</i>, <i>espG</i>, <i>espF</i> and <i>espH</i> respectively. The <i>espZ</i> and <i>tir</i> genes were deleted after the non-LEE effectors, with order 12 and 13 respectively. Scale of 5 kb is indicated at the bottom. <b>B</b>. Effector genes located outside the LEE are localized in integrative elements (IE) and prophages (PP). Effector genes are labeled in red. Pseudogenes are specified with asterisk. The red bars indicate the deletions carried out. The order of deletion is numbered. Scale of 5 kb is indicated at the bottom.</p

EPEC strains used in this work.

<p>EPEC strains used in this work.</p

Translocation of NleC by EPEC effectors mutants induces p65 degradation.

<p><b>A</b>. Western blot detecting NF-κB p65 protein in HeLa cells infected for 4 h (1 h+3 h gentamicin) with EPEC2, EPEC1 and EPEC0 and isogenic strains with <i>nleC</i> integrated in the chromosome. Uninfected (UI) cells and cells infected with WT EPEC are used as controls. Detection of α-tubulin was used as a loading control. <b>B</b>. Quantification of p65 in Hela cells infected with the indicated strains. Protein loading was normalized with α-tubulin. Results are the mean of three independent experiments with standard deviation (SD). One way ANOVA Tukey's Multiple Comparison Test. **<i>p</i> <0.01; ***<i>p</i> <0.001.</p

Functionality of T3SS of the EPEC effector mutants.

<p><b>A</b>. Analysis of secreted and bacterial proteins in the indicated EPEC strains after 4 h of growth in DMEM at 37°C. Top panel: Coomassie staining of proteins found in the extracellular medium labeling the translocators EspABD and the autotransporter EspC. Molecular standards are shown in kDa. Bottom panels: Western blots of bacterial whole-cell protein extracts incubated with polyclonal antibodies to detect EscC, EscJ, EscD injectisome proteins and EspB translocator protein. Detection of cytoplasmic GroEL was used as a loading control. Discontinuous lines indicate borders of independent gels. <b>B</b>. Immunofluorescence confocal microscopy of HeLa cells infected for 90 min with WT EPEC, EPEC2, EPEC1 and EPEC0 strains. EPEC bacteria are labeled with anti-intimin-280 polyclonal (green), actin is labeled with TRITC-phalloidin (red) and cell nuclei are labeled with DAPI (gray). Actin polymerization beneath the adherent bacteria is observed in EPECwt, EPEC2 and EPEC1 strains. Scale bar 5 μm. <b>C.</b> Quantification of the number of HeLa cells with actin pedestals after infection with the indicated strains WT, EPEC2, EPEC1 and EPEC0. The data shown are the mean of two independent experiments with standard deviation (SD). In each experiment, one hundred cells per infection sample were counted. <b>D</b>. Protein translocation into HeLa cells of β-lactamase (Bla) fusion by EPEC effector mutants. HeLa cells were infected for 90 min with the indicated EPEC strains (WT EPEC, EPECΔ<i>escN</i>, EPEC2, EPEC1 EPEC0), expressing EspF_1-20-Bla fusion (WT EPEC, EPECΔ<i>escN</i>, EPEC2, EPEC1, EPEC0) or the control vector pCX340, and then incubated with the BLA substrate CCF2/AM for additional 1 h. Bla activity was quantified measuring the emission ratio of fluorescence at 450/520 nm. Results are the mean of three independent experiments with standard deviation (SD). One way ANOVA Tukey's Multiple Comparison Test. **p<0.01 and ***<i>p</i> <0.001.</p

A/E lesions in human intestinal biopsies infected with EPEC effector mutants.

<p>A/E lesions in human intestinal biopsies infected with EPEC effector mutants.</p