Lymphostatin, a virulence factor of attaching and effacing Escherichia coli, inhibits proliferation and cytokine responses of human T cells in a manner associated with cell cycle arrest but not apoptosis or necrosis.

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.

Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype.

One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1ß, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.

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.

Secretory immunoglobulin A (sIgA) in saliva versus virulence proteins of enteropathogenic Escherichia coli (EPEC) in ill and colonized children.

INTRODUCTION: We evaluated the presence of sIgA in saliva, versus Escherichia coli secreted proteins (Esp) related to the type III secretion system (T3SS), and its semi-quantitative concentration in children under 2 years-old (no longer breastfed) who were previously colonized or infected with enteropathogenic E. coli (EPEC). METHODS: We analyzed the presence of sIgA in 40 children, who previously had positive cultures for EPEC associated (n=17) or not associated (n=23) with diarrhea, using the Western Blot technique versus E. coli secreted proteins: EspABCD. A semi-quantitative measurement of the reaction for each protein was made by its density peaks (OD). RESULTS: We found sIgA versus all or some EspABCD proteins in both groups. However, the ill patients had higher concentrations of these antibodies than colonized patients. DISCUSSION: The presence of sIgA in saliva could reflect an intestinal immune response and their levels could be related to a greater exposure and/or bacterial load.

Salmonella, E. coli, and Citrobacter Type III Secretion System Effector Proteins that Alter Host Innate Immunity.

Bacteria deliver virulence proteins termed 'effectors' to counteract host innate immunity. Protein-protein interactions within the host cell ultimately subvert the generation of an inflammatory response to the infecting pathogen. Here we briefly describe a subset of T3SS effectors produced by enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), Citrobacter rodentium, and Salmonella enterica that inhibit innate immune pathways. These effectors are interesting for structural and mechanistic reasons, as well as for their potential utility in being engineered to treat human autoimmune disorders associated with perturbations in NF-κB signaling.

IL-17 is a protection effector against the adherent-invasive Escherichia coli in murine colitis.

Inflammatory bowel disease (IBD) is caused by aberrant immune responses to the gut microbiota. Among the gut microbiota, adherent-invasive Escherichia Coli (AIEC) is thought to be the pathogen through invading the intestinal epithelial cells and causing inflammation. IL-17 secretion increase, induced by enhanced bacterial adhesion to the intestine epithelium, could on one hand protect the mucosa, but on the other hand, over amount of IL-17 initializes inflammation reactions that in turn damages the mucosa. The relationship between IL-17 and AIEC is still unclear. In this study, we tried to elucidate the function of IL-17 in AIEC-mediated colitis. Wild type (WT) and IL-17 knockout (IL-17 KO) mice were inoculated with AIEC strain E. coli LF82 and treated with dextran sodium sulphate (DSS). Histological examination of the colon was performed. Mucosa damage was assessed and scored. IL-22 and IL-17 in colon tissues were detected by ELISA, qPCR and immunohistochemistry methods. Transient AIEC colonization in IL-17 KO mice resulted in increased intestinal epithelial damage, systemic bacterial burden and mortality compared with WT controls. Moreover, IL-17 is required for the induction of IL-22 in the experimental animal models during AIEC strain E. coli LF82 colonization. These results indicate IL-17 plays a protective role in AIEC strain E. coli LF82 induced colitis by promoting IL-22 secretion.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ / Transferência placentária e colostral de anticorpos reativos a Escherichia coli enteropatogênica com expressão das intiminas α, ß, or γ

Abstract Objective: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. Methods: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, β, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. Results: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. Conclusions: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Resumo Objetivo: As intiminas são adesinas proteicas de Escherichia coli enteropatogênicas (EPEC) e enterro-hemorrágicas (EHEC) capazes de induzir as lesões attaching and effacing nos enterócitos. Anticorpos anti-intiminas são importantes para a proteção contra infecções por EPEC e EHEC porque esses anticorpos inibem a adesão bacteriana e impedem o passo inicial do mecanismo patogênico dessas bactérias. Nós estudamos a transferência de anticorpos maternos anti-intiminas de mães brasileiras saudáveis para os seus recém-nascidos através da placenta e do colostro. Métodos: Anticorpos séricos da classe IgG e secretórios da classe IgA (SIgA) reativos com as porções conservada (cons) e variáveis das intiminas α (vα), β (vβ) e γ (vγ) foram analisados pelo teste de ELISA no sangue e no colostro de 45 parturientes saudáveis e no sangue de cordão umbilical dos seus respectivos recém-nascidos. Resultados: As concentrações de anticorpos reativos com intimina vα foram significativamente mais baixas que as dos anticorpos anti-vγ e anti-cons nas amostras de colostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos, mas as concentrações de anti-cons foram significativamente mais altas tanto nas mães como nos recém-nascidos do que os anticorpos reativos com as regiões variáveis das intiminas. O padrão de transferência de IgG das mães para os recém-nascidos foi muito semelhante para todos os anticorpos anti-intiminas. Os valores de porcentagem de transferência foram semelhantes à transferência de IgG total. Conclusões: Anticorpos anti-intimina são transferidos das mães para os recém-nascidos pela placenta e corroboram a proteção contra infecções por Escherichia coli diarreiogênicas (DEC) conferida pelo aleitamento materno.

Secretion of the Shiga toxin B subunit (Stx1B) via an autotransporter protein optimizes the protective immune response to the antigen expressed in an attenuated E. coli (rEPEC E22Δler) vaccine strain.

We previously developed attenuated rabbit enteropathogenic E. coli (rEPEC) strains which are effective oral vaccines against their parent pathogens by deleting ler, a global regulator of virulence genes. To use these strains as orally administered vectors to deliver other antigens we incorporated the B subunit of shiga-like toxin 1(Stx1) into the passenger domain of the autotransporter EspP expressed on a plasmid. Native EspP enters the periplasm where its passenger domain is exported to the bacterial surface through an outer membrane channel formed by its translocator domain, then cleaved and secreted. Since antigen localization may determine immunogenicity, we engineered derivatives of EspP expressing Stx1B- passenger domain fusions: 1. in cytoplasm 2. in periplasm, 3. surface-attached or 4. secreted. To determine which construct was most immunogenic, rabbits were immunized with attenuated O103 E. coli strain (E22 Δler) alone or expressing Stx1B in each of the above four cellular locations. IgG responses to Stx1B, and toxin-neutralizing antibodies were measured. Animals were challenged with a virulent rabbit Enterohemorrhagic E. coli (EHEC) strain of a different serogroup (O15) than the vaccine strain expressing Stx1 (RDEC-H19) and their clinical course observed. IgG responses to Stx1B subunit were induced in all animals vaccinated with the strain secreting Stx1B, in some vaccinated with surface-expressed Stx1B, but in not animals immunized with periplasmic or cytoplasmic Stx1B. Robust protection was observed only in the group immunized with the vaccine secreting Stx1B. Taken together, our data suggest that secretion of Stx1B, or other antigens, via an autotransporter, may maximize the protective response to live attenuated oral vaccine strains.

Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells.

Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott-Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention.

Stromal Cell-Derived Factor 1 Mediates Immune Cell Attraction upon Urinary Tract Infection.

Urinary tract infection (UTI) is the most common type of bacterial infection in humans. Fifty percent of all women will experience at least one UTI in their lifetime, with uropathogenic Escherichia coli (UPEC) accounting for 80% of reported cases. UTI evokes a complex, well-timed immune response that is crucial for bacterial clearance. The majority of immune cells participating in the immune response are absent from the healthy bladder, and the mechanisms used to recruit them upon UTI are not fully understood. Here, we show that immediately after UPEC infection, bladder epithelial cells secrete stromal cell-derived factor 1 (SDF-1), initiating immune cell accumulation at the site of infection. SDF-1 blockade significantly reduced immune cell migration to the infected bladder, resulting in severe exacerbation of infection. We also show that FimH, the adhesin of type 1 fimbria, one of UPEC's virulence factors, is directly involved in the secretion of SDF-1 upon UTI.

Citrobacter rodentium: a model enteropathogen for understanding the interplay of innate and adaptive components of type 3 immunity.

Citrobacter rodentium is a natural murine intestinal pathogen that shares a core set of virulence factors with the related human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). C. rodentium is now the most widely used small animal model for studying the molecular underpinnings of EPEC and EHEC infections in vivo, including: enterocyte attachment; virulence; colonization resistance; and mucosal immunity. In this review, we discuss type 3 immunity in the context of C. rodentium infection and discuss recent publications that use this model to understand how the innate and adaptive components of immunity intersect to mediate host protection against enteric pathogens and maintain homeostasis with the microbiota.

Innate immunity restricts Citrobacter rodentium A/E pathogenesis initiation to an early window of opportunity.

Citrobacter rodentium infection is a mouse model for the important human diarrheal infection caused by enteropathogenic E. coli (EPEC). The pathogenesis of both species is very similar and depends on their unique ability to form intimately epithelium-adherent microcolonies, also known as "attachment/effacement" (A/E) lesions. These microcolonies must be dynamic and able to self-renew by continuous re-infection of the rapidly regenerating epithelium. It is unknown whether sustained epithelial A/E lesion pathogenesis is achieved through re-infection by planktonic bacteria from the luminal compartment or local spread of sessile bacteria without a planktonic phase. Focusing on the earliest events as C. rodentium becomes established, we show here that all colonic epithelial A/E microcolonies are clonal bacterial populations, and thus depend on local clonal growth to persist. In wild-type mice, microcolonies are established exclusively within the first 18 hours of infection. These early events shape the ongoing intestinal geography and severity of infection despite the continuous presence of phenotypically virulent luminal bacteria. Mechanistically, induced resistance to A/E lesion de-novo formation is mediated by TLR-MyD88/Trif-dependent signaling and is induced specifically by virulent C. rodentium in a virulence gene-dependent manner. Our data demonstrate that the establishment phase of C. rodentium pathogenesis in vivo is restricted to a very short window of opportunity that determines both disease geography and severity.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ.

OBJECTIVE: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. METHODS: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, ß, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. RESULTS: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. CONCLUSIONS: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Linking Pathogen Virulence, Host Immunity and The Microbiota at the Intestinal Barrier.

The mechanisms that allow enteric pathogens to colonize the intestine and host immunity as well as the indigenous microbiota to inhibit pathogen colonization remain poorly understood. Our laboratory is using Citrobacter rodentium, a mouse pathogen that models human infections by enteropathogenic E. coli, to understand the mechanisms that regulate the colonization and clearance of the pathogen in the gut. These studies have revealed how the pathogen colonizes and replicates successfully early during infection and how host immunity and the indigenous microbiota cooperate to eradicate the pathogen in the later stage of the infection. The impairment of the immune system to control the barrier function of the intestine leads to pathogen invasion and the induction of a second layer of host protective immunity to limit the systemic spread of the pathogen.(Presented at the 1931st Meeting, January 17, 2017).

Model of Host-Pathogen Interaction Dynamics Links In Vivo Optical Imaging and Immune Responses.

Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells.

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1ß and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11-IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.

The Genetics of Enteropathogenic Escherichia coli Virulence.

In many parts of the world, enteropathogenic Escherichia coli (EPEC) are a leading cause of death in children with diarrhea. Much of what we know about the pathogenesis of EPEC infections is based on the study of one or two prototypic strains that have provided deep insight into the precise mechanisms by which EPEC colonizes the intestine, evades host immunity, and spreads from person to person. In some cases, defining the biochemical activity of the host-interacting effector proteins from these prototypic strains has led to the discovery of novel post-translational protein modifications and new understandings of biology and host-pathogen interactions. However, genomic analysis of recent EPEC isolates has revealed that the EPEC pathotype is more diverse than previously appreciated. Although by definition all strains carry the locus of enterocyte effacement, the effector repertoires of different clonal groups are quite divergent, suggesting that there is still a great deal to learn about the genetic basis of EPEC virulence.

Modulation of the Inflammasome Signaling Pathway by Enteropathogenic and Enterohemorrhagic Escherichia coli.

Innate immunity is an essential component in the protection of a host against pathogens. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are known to modulate the innate immune responses of infected cells. The interference is dependent on their type III secretion system (T3SS) and T3SS-dependent effector proteins. Furthermore, these cytosolically injected effectors have been demonstrated to engage multiple immune signaling pathways, including the IFN/STAT, MAPK, NF-κB, and inflammasome pathways. In this review, recent work describing the interaction between EPEC/EHEC and the inflammasome pathway will be discussed.