SUMMARY The airway epithelium acts as a frontline defense against respiratory viruses, not only as a physical barrier and through the mucociliary apparatus but also through its immunological functions. It initiates multiple innate and adaptive immune mechanisms which are crucial for efficient antiviral responses. The interaction between respiratory viruses and airway epithelial cells results in production of antiviral substances, including type I and III interferons, lactoferrin, β-defensins, and nitric oxide, and also in production of cytokines and chemokines, which recruit inflammatory cells and influence adaptive immunity. These defense mechanisms usually result in rapid virus clearance. However, respiratory viruses elaborate strategies to evade antiviral mechanisms and immune responses. They may disrupt epithelial integrity through cytotoxic effects, increasing paracellular permeability and damaging epithelial repair mechanisms. In addition, they can interfere with immune responses by blocking interferon pathways and by subverting protective inflammatory responses toward detrimental ones. Finally, by inducing overt mucus secretion and mucostasis and by paving the way for bacterial infections, they favor lung damage and further impair host antiviral mechanisms.

Deficient type I interferon-β and type III interferon-λ induction by rhinoviruses has previously been reported in mild/moderate atopic asthmatic adults. No studies have yet investigated if this occurs in severe therapy resistant asthma (STRA). Here, we show that compared with non-allergic healthy control children, bronchial epithelial cells cultured ex vivo from severe therapy resistant atopic asthmatic children have profoundly impaired interferon-β and interferon-λ mRNA and protein in response to rhinovirus (RV) and polyIC stimulation. Severe treatment resistant asthmatics also exhibited increased virus load, which negatively correlated with interferon mRNA levels. Furthermore, uninfected cells from severe therapy resistant asthmatic children showed lower levels of Toll-like receptor-3 mRNA and reduced retinoic acid inducible gene and melanoma differentiation-associated gene 5 mRNA after RV stimulation. These data expand on the original work, suggesting that the innate anti-viral response to RVs is impaired in asthmatic tissues and demonstrate that this is a feature of STRA.

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Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx 2 variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx 2-vhb variant, which was frequently associated with stx 2 , stx 2-vha or stx 2c . Levels of stx 2 and stx 2 -related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx 2 variant than in the remaining strains of seropathotype C. The stx 2-vhb genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx 2 -related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx 2 -related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx 2 gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.

Shiga-toxin (Stx) is the cardinal virulence factor of enterohemorrhagic Escherichia coli (EHEC). The genes encoding Stx are carried by a lambdoid phage integrated in the bacterial genome and are fully expressed after a bacterial SOS response induced by DNAdamaging agents. Because nitric oxide (NO) is an essential mediator of the innate immune response of infected colonic mucosa, we aimed to determine its role in Stx production by EHEC. Here we demonstrate that chemical or cellular sources of NO inhibit spontaneous and mitomycin C-induced stx mRNA expression and Stx synthesis, without altering EHEC viability. The synthesis of stx phage is also reduced by NO. This inhibitory effect apparently occurs through the NO-mediated sensitization of EHEC because mutation of the NO sensor nitrite-sensitive repressor results in loss of NO inhibiting activity on stx expression. Thus our findings identify NO as an inhibitor of stx expressing-phage propagation and Stx release and thus as a potential protective factor limiting the development of hemolytic syndromes.bacterial infection ͉ mucosal immunology E nterohemorrhagic Escherichia coli (EHEC) are pathogens carried by healthy rearing animals. After infection through the ingestion of contaminated food, EHEC colonize the large intestine and cause gastrointestinal diseases ranging from uncomplicated diarrhea to hemorrhagic colitis. Life-threatening complications, such as hemolytic-uremic syndrome (HUS), develop in Ϸ5-10% of EHEC-infected patients. HUS is defined by a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, which can yield to a chronic renal failure and even death (1-3). O157:H7 is the main EHEC serotype implicated in HUS in Europe and North America (3). The few recent large outbreaks (4, 5) underline that prevention of primary infection in human remains an elusive goal. Therefore, understanding host-EHEC interactions remains a critical issue in fighting bacterial infection and HUS development.The main EHEC virulence factor associated with severe human diseases is the Shiga toxin (Stx). Both Stx1 and Stx2 are heteropolymers constituted by a catalytic A subunit and five B subunits implicated in the binding to the receptor glycolipid globotriaosylceramide-3 of endothelial cells. Internalized Stx alters ribosomal function and induces the death of vascular cells (1, 3). Stx1 and Stx2 are encoded by two type lysogenic phages integrated in the bacterial genome (6). In a lysogen, the expression of the phage operons is controlled by the protein CI. As a result of EHEC exposure to DNA-damaging agents such as mitomycin C (7) or H 2 O 2 (8), RecA, which is part of the so-called SOS response, is activated by single-strand DNA and promotes the autocleavage of CI (9). Then, a regulatory cascade yields to the respective expression of the genes encoding the antiterminators N and Q, Stx, and proteins of phage morphogenesis and lysis (9-13). Bacteria are lysed and release Stx and free phage particles in the medium.An important hallmark of EHEC pathoge...

Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx 2 mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx 2 repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

Expression of genes of the locus of enterocyte effacement (LEE) is essential for adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelial cells. Gut factors that may modulate LEE gene expression may therefore influence the outcome of the infection. Because nitric oxide (NO) is a critical effector of the intestinal immune response that may induce transcriptional regulation in enterobacteria, we investigated its influence on LEE expression in EHEC O157:H7. We demonstrate that NO inhibits the expression of genes belonging to LEE1, LEE4, and LEE5 operons, and that the NO sensor nitrite-sensitive repressor (NsrR) is a positive regulator of these operons by interacting directly with the RNA polymerase complex. In the presence of NO, NsrR detaches from the LEE1/4/5 promoter regions and does not activate transcription. In parallel, two regulators of the acid resistance pathway, GadE and GadX, are induced by NO through an indirect NsrR-dependent mechanism. In this context, we show that the NO-dependent LEE1 down-regulation is due to absence of NsrR-mediated activation and to the repressor effect of GadX. Moreover, the inhibition of expression of LEE4 and LEE5 by NO is due to loss of NsrR-mediated activation, to LEE1 down-regulation and to GadE up-regulation. Lastly, we establish that chemical or cellular sources of NO inhibit the adherence of EHEC to human intestinal epithelial cells. These results highlight the critical effect of NsrR in the regulation of the LEE pathogenicity island and the potential role of NO in the limitation of colonization by EHEC.

Enterohemorrhagic Escherichia coli (EHEC) are the causative agent of hemolytic-uremic syndrome. In the first stage of the infection, EHEC interact with human enterocytes to modulate the innate immune response. Inducible NO synthase (iNOS)-derived NO is a critical mediator of the inflammatory response of the infected intestinal mucosa. We therefore aimed to analyze the role of EHEC on iNOS induction in human epithelial cell lines. In this regard, we show that EHEC down-regulate IFN-γ-induced iNOS mRNA expression and NO production in Hct-8, Caco-2, and T84 cells. This inhibitory effect occurs through the decrease of STAT-1 activation. In parallel, we demonstrate that EHEC stimulate the rapid inducible expression of the gene hmox-1 that encodes for the enzyme heme oxygenase-1 (HO-1). Knock-down of hmox-1 gene expression by small interfering RNA or the blockade of HO-1 activity by zinc protoporphyrin IX abrogated the EHEC-dependent inhibition of STAT-1 activation and iNOS mRNA expression in activated human enterocytes. These results highlight a new strategy elaborated by EHEC to control the host innate immune response.