Generation of enteropathogenic E. coli strains lacking the repertoire of effectors translocated by the type III protein secretion system and their characterization in the infection of cultured cell lines and human intestinal biopsies

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-10-2016Esta tesis tiene embargado el acceso al texto completo hasta el 26-04-2018Although most Escherichia coli isolates are harmless commensals of the gastrointestinal tract, some strains have acquired specific virulence factors, like pathogenicity islands, insertion elements (IEs), and prophages (PPs), to become highly adapted pathogens. The enteropathogenic E. coli (EPEC) is an important category of diarrheagenic bacteria causing acute and chronic diarrhea in infants. The hallmark of EPEC infection is the formation of attachment and effacement (A/E) lesion in the intestinal mucosa surface, which is characterized by the intimate attachment of the bacteria to the enterocyte, microvilli effacement, and the formation of actin-pedestal-like structures underneath the attached bacteria. EPEC is endowed of a 35 kb pathogenicity island called the locus of enterocyte effacement (LEE) that contains all the genes necessary for the assembly of a type III secretion system (T3SS) injectisome. Through these injectisome EPEC translocates multiple effector proteins into the host cell to subvert cellular functions in benefit of the infection. The prototype strain E2348/69 of EPEC O127:H6 is endowed of six LEE encoded effectors and 17 non-LEE encoded effectors. We have engineered a set of effector mutant EPEC strains using suicide vectors to delete the whole repertoire of effector genes of this prototype EPEC strain. Genome manipulation did not affect the functionality of the T3SS injectisome. The deletion strategy was based on suicide or termosensitive plasmid integration by homologous recombination and the markerless resolution of co-integrants after I-SceI digestion. We did markerless integration of map, espH and nleC in their original locus in EPEC2 (maintain EspZ and Tir), EPEC1 (maintaining only Tir) and EPEC0 (effector-less) mutant strains. These strains were able to translocate functional effectors from chromosomal expression into HeLa cells. We infected intestinal human biopsies with the effector mutant EPEC strains to identify the effectors necessary for the induction of the A/E lesion in human intestinal tissues. We found that while EPEC2 and EPEC1 mutant strains were able to induce the actin-pedestal formation in HeLa cells in vitro, none of the biopsies infected with these strains had A/E lesion. These results demonstrated that effectors besides Tir and EspZ are essential to induce the A/E lesion formation in intestinal biopsies. We infected intestinal biopsies with several effector mutant EPEC strains and we found that effectors located outside the LEE are essential to induce efficient A/E lesion on human intestinal biopsies. Additionally we found that non-LEE effectors are characterized by having an additive effect to allow the A/E lesion development in these intestinal surfaces and that Efa1/LifA homologous proteins seem to play a major role in this process. Our results with intestinal biopsies strongly suggest that non-LEE effectors are necessary for the efficient formation of A/E lesion in the in vivo situation.Aunque la mayoría de los aislados de Escherichia coli son comensales del tracto gastrointestinal, algunas cepas han adquirido factores de virulencia específicos, como islas de patogenicidad, elementos integrativos (IEs), y profagos (PPs), para convertirse en patógenos altamente adaptados. La cepas de Escherichia coli enteropatógena (EPEC) son una categoría importante de bacterias productoras de diarrea aguda y crónica en niños de corta edad. La característica distintiva de la infección por EPEC es la formación de la lesión llamada de unión y borrado A/E (Attaching and Effacing lesion), que se caracteriza por una unión intima de la bacteria a los enterocitos, la destrucción de las microvellosidades, y la formación de estructuras en forma de pedestales debajo de las bacterias unidas. EPEC está dotada de una isla de patogenicidad de 35 kb llamada LEE (locus of enterocyte effacement) que contiene todos los genes necesarios para ensamblar los inyectisomas del sistema de secreción tipo III (T3SS). A través de estos inyectisomas EPEC transloca proteínas efectoras a la célula huésped para manipular diversas funciones celulares en beneficio de la infección. La cepa prototipo E2348/69 de EPEC O127:H6 tiene seis efectores codificados en la isla LEE y 17 efectores codificados fuera de la isla LEE, llamados genéricamente efectores no-LEE. Hemos construido un grupo de cepas mutantes en efectores de EPEC, utilizando vectores suicidadas para delecionar el repertorio de efectores de la cepa prototipo. La manipulación genómica no afectó la funcionalidad de los inyectisomas del T3SS. La estrategia seguida se basa en el uso de plásmidos suicidas y termosensibles que se integran por recombinación homóloga, seguida de una resolución de los co-integrantes tras digestión con I-SceI, produciendo deleciones libres de marcadores. También hemos realizado una integración libre de marcas de los genes map, espH y nleC en su sitio original en el cromosoma de las cepas mutantes EPEC2 (mantiene EspZ y Tir), EPEC1 (mantiene Tir) y EPEC0 (sin efectores). Las cepas generadas son capaces de translocar desde su expresión cromosómica los efectores individuales de forma funcional a células HeLa. Infectamos biopsias intestinales humanas con las cepas de EPEC mutantes en efectores para identificar los efectores necesarios para inducir la formación de la lesión A/E en tejidos intestinales humanos. Identificamos que mientras las cepas mutantes EPEC2 y EPEC1 inducen la formación de pedestales de actina durante la infección in vitro de células HeLa, ninguna de las biopsias infectadas por estas cepas presentó lesiones A/E. Estos resultados demuestran que otros efectores además de Tir y EspZ son esenciales para inducir la formación de la lesión A/E en las biopsias intestinales. Infectamos biopsias intestinales con varias cepas de EPEC mutantes en efectores y descubrimos que los efectores localizados fuera de la isla LEE son esenciales para inducir eficientemente la lesión A/E en las biopsias intestinales humanas. Además demostramos que los efectores no-LEE se caracterizan por tener un efecto aditivo para permitir el desarrollo de la lesión A/E en estas superficies intestinales y que las proteínas homologas a Efa1/LifA parecen jugar un papel principal en este proceso. Nuestros resultados con biopsias intestinales apoyan un papel de los efectores no-LEE en la formación eficiente de la lesión A/E en la situación in vivo.Este trabajo ha sido realizado en el Departamento de Biotecnología Microbiana del Centro Nacional de Biotecnología del Consejo Superior de Investigaciones Científicas (CNB-CSIC), gracias a un contrato predoctoral JAE del CSIC y a los fondos de investigación de los proyectos del Ministerio de Economía y Competitividad (BIO2011-26689) del Gobierno de España y del European Research Council (ERC-2012-ADG_20120314)

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

Systematic deletion of type III secretion system effectors in enteropathogenic E. coli unveils the role of non-LEE effectors in A/E lesion formation

Enteropathogenic E. coli (EPEC) is a diarrheagenic human pathogen. The hallmark of EPEC infection is the formation of the attachment and effacement (A/E) lesion in intestinal epithelial cells, characterized by the effacement of brush border microvilli and the intimate bacterial attachment to the enterocyte in actin-rich pedestal-like structures. The locus of enterocyte effacement (LEE) in the EPEC genome encodes a type III protein secretion system (T3SS) that translocates multiple effector proteins into the host cell to subvert cellular functions for the benefit of the pathogen. These effectors are encoded both within the LEE and outside the LEE. In vitro cell culture infections have shown that LEE effectors are required for intimate bacterial attachment to epithelial cells whereas non-LEE effectors mostly play a role in modulating inflammation and cell apoptosis in the gut epithelium. We constructed a set of EPEC mutant strains harbouringving deletions in the complete repertoire of genes encoding T3SS-effectors. Infection of human intestinal in vitro organ cultures (IVOC) with these mutant strains surprisingly revealed that non-LEE effectors are also needed to induce efficient A/E lesion formation in intestinal mucosal tissue

Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx–dependent pyroptosis in intestinal epithelial cells

© 2020 Zhong et al.Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.T.I.R and J.S.C are supported by the Cancer Research UK Centre grant C309/A25144. GF is supported a Wellcome Investigator Award 107057/Z/15/Z. QZ is supported by Imperial College President’s PhD Scholarship. GF is supported by a Wellcome Investigator Award (107057/Z/15/Z). T.I.R and J.S.C were funded by the CRUK Centre grant (C309/A25144)

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

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

EPEC strains used in this work.

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

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

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

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