Enteropathogenic Escherichia coli modulates the virulence and pathogenicity of Entamoeba dispar.

Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.

Colonization of the gut mucosa of colorectal cancer patients by pathogenic mucosa-associated Escherichia coli strains.

Some strains of Escherichia coli are known to be involved in the pathogenesis of colorectal cancer (CRC). The aim of current study was to compare the general characteristics of the E. coli from CRC patients and healthy participants. A total of 96 biopsy samples from 48 CRC patients and 48 healthy participants, were studied. The clonality of the E. coli isolates was analyzed by Enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR) method. The strains were tested by PCR to determine the prevalence of different virulence factors. According to the results of ERIC-PCR analysis, (from the 860 E. coli isolates) 60 strains from CRC patients and 41 strains from healthy controls were identified. Interestingly, the majority of the strains of both groups were in the same cluster. Enteropathogenic E. coli (EPEC) was detected significantly more often in CRC patients (21.6 %) than in healthy participants (2.4 %) (p < 0.05). The Enteroaggregative E. coli (EAEC) was found in 18.33 % of the strains of CRC patients. However, other pathotypes were not found in the E. coli strains of both groups. Furthermore, all the studied genes encoding for virulence factors seemed to be more prevalent in the strains belonging to CRC patients. Among the virulence genes, the statistical difference regarding the frequency of fuyA, chuA, vat, papC, hlyA and cnf1 genes was found significant (p < 0.05). In conclusion, E. coli strains that carry extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC) multiple virulence factors colonize the gut mucosa of CRC patients.

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli.

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Bacterial Genotoxin Accelerates Transient Infection-Driven Murine Colon Tumorigenesis.

Chronic and low-grade inflammation associated with persistent bacterial infections has been linked to colon tumor development; however, the impact of transient and self-limited infections in bacterially driven colon tumorigenesis has remained enigmatic. Here we report that UshA is a novel genotoxin in attaching/effacing (A/E) pathogens, which include the human pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and their murine equivalent Citrobacter rodentium (CR). UshA harbors direct DNA digestion activity with a catalytic histidine-aspartic acid dyad. Injected via the type III secretion system (T3SS) into host cells, UshA triggers DNA damage and initiates tumorigenic transformation during infections in vitro and in vivo. Moreover, UshA plays an indispensable role in CR infection-accelerated colon tumorigenesis in genetically susceptible Apc MinΔ716/+ mice. Collectively, our results reveal that UshA, functioning as a bacterial T3SS-dependent genotoxin, plays a critical role in prompting transient and noninvasive bacterial infection-accelerated colon tumorigenesis in mice. SIGNIFICANCE: We identified UshA, a novel T3SS-dependent genotoxin in A/E pathogens that possesses direct DNA digestion activity and confers bacterially accelerated colon tumorigenesis in mice. Our results demonstrate that acute and noninvasive infection with A/E pathogens harbors a far-reaching impact on the development of colon cancer.This article is highlighted in the In This Issue feature, p. 1.

Evolutionary Dynamics Based on Comparative Genomics of Pathogenic Escherichia coli Lineages Harboring Polyketide Synthase (pks) Island.

The genotoxin colibactin is a secondary metabolite produced by the polyketide synthase (pks) island harbored by extraintestinal pathogenic E. coli (ExPEC) and other members of the Enterobacteriaceae that has been increasingly reported to have critical implications in human health. The present study entails a high-throughput whole-genome comparison and phylogenetic analysis of such pathogenic E. coli isolates to gain insights into the patterns of distribution, horizontal transmission, and evolution of the island. For the current study, 23 pks-positive ExPEC genomes were newly sequenced, and their virulome and resistome profiles indicated a preponderance of virulence encoding genes and a reduced number of genes for antimicrobial resistance. In addition, 4,090 E. coli genomes from the public domain were also analyzed for large-scale screening for pks-positive genomes, out of which a total of 530 pks-positive genomes were studied to understand the subtype-based distribution pattern(s). The pks island showed a significant association with the B2 phylogroup (82.2%) and a high prevalence in sequence type 73 (ST73; n = 179) and ST95 (n = 110) and the O6:H1 (n = 110) serotype. Maximum-likelihood (ML) phylogeny of the core genome and intergenic regions (IGRs) of the ST95 model data set, which was selected because it had both pks-positive and pks-negative genomes, displayed clustering in relation to their carriage of the pks island. Prevalence patterns of genes encoding RM systems in the pks-positive and pks-negative genomes were also analyzed to determine their potential role in pks island acquisition and the maintenance capability of the genomes. Further, the maximum-likelihood phylogeny based on the core genome and pks island sequences from 247 genomes with an intact pks island demonstrated horizontal gene transfer of the island across sequence types and serotypes, with few exceptions. This study vitally contributes to understanding of the lineages and subtypes that have a higher propensity to harbor the pks island-encoded genotoxin with possible clinical implications.IMPORTANCE Extraintestinal pathologies caused by highly virulent strains of E. coli amount to clinical implications with high morbidity and mortality rates. Pathogenic E. coli strains are evolving with the horizontal acquisition of mobile genetic elements, including pathogenicity islands such as the pks island, which produces the genotoxin colibactin, resulting in severe clinical outcomes, including colorectal cancer progression. The current study encompasses high-throughput comparative genomics and phylogenetic analyses to address the questions pertaining to the acquisition and evolution pattern of the genomic island in different E. coli subtypes. It is crucial to gain insights into the distribution, transfer, and maintenance of pathogenic islands, as they harbor multiple virulence genes involved in pathogenesis and clinical implications of the infection.

Measuring Effector-Mediated Modulation of Inflammatory Responses to Infection with Enteropathogenic and Shiga Toxin-Producing E. coli.

Shiga toxin-producing Escherichia coli (STEC) and the related pathogen enteropathogenic Escherichia coli (EPEC) use a type III secretion system to translocate effector proteins into host cells to modulate inflammatory signaling pathways during infection. Here we describe the procedures to investigate effector-driven modulation of host inflammatory signaling pathways in mammalian cells where bacterial effectors are ectopically expressed or in cell lines infected with STEC or EPEC. We focus on the TNF-induced NF-κB response by examining IκBα degradation by immunoblot and p65 nuclear localization in addition to using an NF-κB-dependent luciferase reporter and cytokine secretion assays. These methods can be adapted for examining effector-mediated modulation of other inflammatory stimuli and host signaling pathways.

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

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.

Quantitative analysis and virulence phenotypes of atypical enteropathogenic Escherichia coli (EPEC) acquired from diarrheal stool samples from a Midwest US hospital.

Infectious diarrhea causes approximately 179 million illnesses annually in the US. Multiplex PCR assays for enteric pathogens detect enteropathogenic Escherichia coli (EPEC) in 12-29% of diarrheal stool samples from all age groups in developed nations. The aim of this study was to isolate and characterize EPEC from diarrhea samples identified as EPEC positive by BioFire Gastrointestinal Panel (GIP). EPEC is the second most common GIP-detected pathogen, equally present in sole and mixed infections peaking during summer months. EPEC bacterial load is higher in samples with additional pathogens. EPEC-GIP-positive stool samples were cultured on MacConkey II agar and analyzed by colony PCR for eaeA and bfpA to identify and classify EPEC isolates as typical (tEPEC) or atypical (aEPEC). EPEC were not recovered from the majority of stool samples with only 61 isolates obtained from 277 samples; most were aEPEC from adults. bfpA-mRNA was severely diminished in 3 of 4 bfpA-positive isolates. HeLa and SKCO-15 epithelial cells were infected with EPEC isolates and virulence-associated phenotypes, including adherence pattern, attachment level, pedestal formation, and tight junction disruption, were assessed. All aEPEC adherence patterns were represented with diffuse adherence predominating. Attachment rates of isolates adhering with defined adherence patterns were higher than tEPEC lacking bfpA (ΔbfpA). The majority of isolates formpedestals. All but one isolate initially increases but ultimately decreases transepithelial electrical resistance of SKCO-15 monolayers, similar to ΔbfpA. Most isolates severely disrupt occludin; ZO-1 disruption is variable. Most aEPEC isolates induce more robust virulence-phenotypes in vitro than ΔbfpA, but less than tEPEC-E2348/69.

Simulated microgravity suppresses MAPK pathway-mediated innate immune response to bacterial infection and induces gut microbiota dysbiosis.

During spaceflight, astronauts are subjected to various physical stressors including microgravity, which could cause immune dysfunction and thus potentially predispose astronauts to infections and illness. However, the mechanisms by which microgravity affects innate immunity remain largely unclear. In this study, we conducted RNA-sequencing analysis to show that simulated microgravity (SMG) suppresses the production of inflammatory cytokines including tumor necrosis factor (TNF) and interleukin-6 (IL-6) as well as the activation of the innate immune signaling pathways including the p38 mitogen-activated protein kinase (MAPK) and the Erk1/2 MAPK pathways in the Enteropathogenic escherichia coli (EPEC)-infected macrophage cells. We then adopted hindlimb-unloading (HU) mice, a model mimicking the microgravity of a spaceflight environment, to demonstrate that microgravity suppresses proinflammatory cytokine-mediated intestinal immunity to Citrobacter rodentium infection and induces the disturbance of gut microbiota, both of which phenotypes could be largely corrected by the introduction of VSL#3, a high-concentration probiotic preparation of eight live freeze-dried bacterial species. Taken together, our study provides new insights into microgravity-mediated innate immune suppression and intestinal microbiota disturbance, and suggests that probiotic VSL#3 has great potential as a dietary supplement in protecting individuals from spaceflight mission-associated infections and gut microbiota dysbiosis.

ß-arrestin 2 quenches TLR signaling to facilitate the immune evasion of EPEC.

The protein translocated intimin receptor (Tir) from enteropathogenic Escherichia coli shares sequence similarity with the host cellular immunoreceptor tyrosine-based inhibition motifs (ITIMs). The ITIMs of Tir are required for Tir-mediated immune inhibition and evasion of host immune responses. However, the underlying molecular mechanism by which Tir regulates immune inhibition remains unclear. Here we demonstrated that ß-arrestin 2, which is involved in the G-protein-coupled receptor (GPCR) signal pathway, interacted with Tir in an ITIM-dependent manner. For the molecular mechanism, we found that ß-arrestin 2 enhanced the recruitment of SHP-1 to Tir. The recruited SHP-1 inhibited K63-linked ubiquitination of TRAF6 by dephosphorylating TRAF6 at Tyr288, and inhibited K63-linked ubiquitination and phosphorylation of TAK1 by dephosphorylating TAK1 at Tyr206, which cut off the downstream signal transduction and subsequent cytokine production. Moreover, the inhibitory effect of Tir on immune responses was diminished in ß-arrestin 2-deficient mice and macrophages. These findings suggest that ß-arrestin 2 is a key regulator in Tir-mediated immune evasion, which could serve as a new therapeutic target for bacterial infectious diseases.

Adherent-Invasive E. coli: Update on the Lifestyle of a Troublemaker in Crohn's Disease.

Besides genetic polymorphisms and environmental factors, the intestinal microbiota is an important factor in the etiology of Crohn's disease (CD). Among microbiota alterations, a particular pathotype of Escherichia coli involved in the pathogenesis of CD abnormally colonizes the intestinal mucosa of patients: the adherent-invasive Escherichia coli (AIEC) pathobiont bacteria, which have the abilities to adhere to and to invade intestinal epithelial cells (IECs), as well as to survive and replicate within macrophages. AIEC have been the subject of many studies in recent years to unveil some genes linked to AIEC virulence and to understand the impact of AIEC infection on the gut and consequently their involvement in CD. In this review, we describe the lifestyle of AIEC bacteria within the intestine, from the interaction with intestinal epithelial and immune cells with an emphasis on environmental and genetic factors favoring their implantation, to their lifestyle in the intestinal lumen. Finally, we discuss AIEC-targeting strategies such as the use of FimH antagonists, bacteriophages, or antibiotics, which could constitute therapeutic options to prevent and limit AIEC colonization in CD patients.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Efectos del medio de cultivo y de la metodología aplicada sobre la formación de biopelículas de 2 cepas de Escherichia coli diarreagénicas / Effects of the culture medium and the methodology applied on the biofilm formation of 2 diarrheagenic Escherichia coli strains

La capacidad de formar biopelículas de los microorganismos patógenos en gran variedad de ambientes, superficies y condiciones trae consigo un importante riesgo, tanto para la industria alimentaria como para la salud pública. Este trabajo tuvo como objetivo evaluar y comparar los efectos de la metodología empleada y de los medios de cultivo utilizados, sobre la capacidad de una cepa de Escherichia coli verotoxigénica no O157 y una enteropatogénica de formar biopelículas sobre una superficie de poliestireno. Se ensayaron 2 variantes metodológicas en cultivo estático y se utilizaron medios de cultivo con diferente composición. Los resultados mostraron que ambas cepas formaron una mayor cantidad de biopelícula en cultivo en LB suplementado con glucosa, con recambio del medio a las 24 h y la cuantificación de la biopelícula realizada a las 48 h de incubación. Dichas condiciones podrían ser utilizadas en futuros estudios sobre formación de biopelícula.

The ability to form biofilms of pathogenic microorganisms in a wide variety of environments, surfaces and conditions constitute an important risk, both for the food industry and for public health. The aim of this work was to evaluate and to compare the effects of the methodology applied and the culture medium used on the ability of a non-O157 verotoxigenic Escherichia coli strain and an enteropathogenic strain to form biofilm on polystyrene surface. Two methodological variants were tested in static culture and culture mediums with different composition were used. The results showed that both strains were able to form a greater biofilm under culture in LB supplemented with glucose, with medium replacement at 24 h and the quantification of the biofilm carried out at 48 h of incubation. These conditions could be used in future studies on biofilm formation.

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages.

Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains.

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

Regulation of virulence and motility by acetate in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli colonizes the human small intestine and causes severe diarrhea. Short-chain fatty acids are abundant in the intestine owing to the metabolic activity of the microflora and are important for intestinal health. Here, we found that acetate promotes the adherence of enteropathogenic E. coli O127:H6 to Caco-2 intestinal epithelial cells and its motility on semi-solid Luria-Bertani agar by activating the expression of locus of enterocyte effacement genes and flagellar genes, respectively. The effect of acetate on locus of enterocyte effacement gene expression is mediated by Ler, the master regulator of locus of enterocyte effacement genes, whereas the regulation of flagellar genes by acetate is dependent on the RNA polymerase sigma factor FliA. Conversely, formate, propionate, and butyrate had little or no effect on enteropathogenic E. coli O127:H6 adherence and motility. Finally, the acetate-mediated regulatory pathway was found to be a widespread mechanism used by a range of enteropathogenic E. coli to mediate bacterial virulence and motility. Therefore, upon entering the human small intestine, enteropathogenic E. coli may respond to the higher acetate level to enhance its virulence and motility, leading to efficient colonization of the target niche.

Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells.

Enteropathogenic Escherichia coli (EPEC) subvert host cell signaling pathways by injecting effector proteins via a Type 3 Secretion System (T3SS). The T3SS-dependent EspB protein is a multi-functional effector protein, which contributes to adherence and translocator pore formation and after injection exhibits several intracellular activities. In addition, EspB is also secreted into the environment. Effects of secreted EspB have not been reported thus far. As a surrogate for secreted EspB we employed recombinant EspB (rEspB) derived from the prototype EPEC strain E2348/69 and investigated the interactions of the purified protein with different human epithelial and immune cells including monocytic THP-1 cells, macrophages, dendritic cells, U-937, epithelial T84, Caco-2, and HeLa cells. To assess whether these proteins might exert a cytotoxic effect we monitored the release of lactate dehydrogenase (LDH) as well as propidium iodide (PI) uptake. For comparison, we also investigated several homologs of EspB such as IpaD of Shigella, and SipC, SipD, SseB, and SseD of Salmonella as purified recombinant proteins. Interestingly, cytotoxicity was only observed in THP-1 cells and macrophages, whereas epithelial cells remained unaffected. Cell fractionation and immune fluorescence experiments showed that rEspB enters cells autonomously, which suggests that EspB might qualify as a novel cell-penetrating effector protein (CPE). Using specific organelle tracers and inhibitors of signaling pathways we found that rEspB destroys the mitochondrial membrane potential - an indication of programmed cell death induction in THP-1 cells. Here we show that EspB not only constitutes an essential part of the T3SS-nanomachine and contributes to the arsenal of injected effector proteins but, furthermore, that secreted (recombinant) EspB autonomously enters host cells and selectively induces cell death in immune cells.

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

Escherichia coli type III secretion system 2 regulator EtrA promotes virulence of avian pathogenic Escherichia coli.

The Escherichia coli type III secretion system 2 (ETT2) is found in most E. coli strains, including pathogenic and commensal strains. Although many ETT2 gene clusters carry multiple genetic mutations or deletions, ETT2 is known to be involved in bacterial virulence. In enterohaemorrhagic E. coli (EHEC), ETT2 affects adhesion through the regulator EtrA, which regulates transcription and secretion of the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). To date, no studies have been conducted on the role of EtrA in the virulence of avian pathogenic E. coli (APEC), which harbours only ETT2. Thus, we constructed etrA mutant and complemented strains of APEC and evaluated their phenotypes and pathogenicities. We found that the etrA gene deletion significantly reduced bacterial survival in macrophages, and proliferation and virulence in ducks. In addition, the etrA gene deletion reduced expression of the APEC fimbriae genes. Upregulation of genes encoding the pro-inflammatory cytokines interleukin (IL)-1ß and IL-8 was also observed in HD-11 macrophages infected with the etrA gene mutant strain compared to the wild-type strain. Furthermore, the altered capacities of the mutant strain were restored by genetic complementation. Our observations demonstrate that the ETT2 regulator EtrA contributes to the virulence of APEC.