The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC.
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1β. EPEC and EHEC are known to elicit inflammasome activation in macrophages and epithelial cells; however, whether the pathogens actively counteract such innate immune responses is unknown. Using a series of compound effector-gene deletion strains of EPEC, we screened and identified NleA, which could subdue host IL-1β secretion. It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA. Immunostaining of human macrophage-like cells following infection revealed limited formation of inflammasome foci with constituents of total caspase-1, ASC and NLRP3 in the presence of NleA. Pulldown of PMA-induced differentiated THP-1 lysate with purified MBP-NleA reveals that NLRP3 is a target of NleA. The interaction was verified by an immunoprecipitation assay and direct interaction assay in which purified MBP-NleA and GST-NLRP3 were used. We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains. Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation. Cumulatively, our findings provide the first example of EPEC-mediated suppression of inflammasome activity in which NieA plays a novel role in controlling the host immune response through targeting of NLRP3.
GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.
SummaryEnterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea and other severe symptoms such as haemorrhagic uraemic syndrome. The expression of virulence genes on the locus for enterocyte effacement (LEE) and associated genes is regulated by a variety of factors, including transcriptional regulators and environmental signals. Butyrate, one of the major short-chain fatty acids present in the intestine, enhances expression of LEE genes and flagella biosynthesis genes in EHEC O157:H7, resulting in increased bacterial adherence and motility. Here, we show that expression of the leuO gene, which encodes a LysR-type transcriptional regulator, is enhanced by butyrate via Lrp, which is also necessary for butyrateinduced responses of LEE genes. LeuO expression induces prolonged activation of the promoter of LEE1 operon, including the ler gene, as well as virulence mechanisms such as microcolony formation. Activation of the LEE1 promoter by LeuO depends on another regulator, called Pch. The response of the leuO promoter to butyrate requires two virulence regulators, Pch and Ler, in addition to Lrp. Pch, Ler and Lrp bind the upstream region of the leuO promoter. Thus, leuO is involved in butyrate-enhanced expression of LEE genes through a positive feedback mechanism, but its expression and action on the LEE1 promoter are dependent on the virulence regulators Pch and Ler.
Antimicrobial peptides (AMPs) are important components of the innate immune system. Enterohaemorrhagic Escherichia coli (EHEC), a food-borne pathogen causing serious diarrheal diseases, must overcome attack by AMPs. Here, we show that resistance of EHEC against human cathelicidin LL-37, a primary AMP, was enhanced by butyrate, which has been shown to act as a stimulant for the expression of virulence genes. The increase of resistance depended on the activation of the ompT gene, which encodes the outer membrane protease OmpT for LL-37. The expression of the ompT gene was enhanced through the activation system for virulence genes. The increase in ompT expression did not result in an increase in OmpT protease in bacteria but in enhancement of the production of OmpT-loaded outer membrane vesicles (OMVs), which primarily contributed to the increase in LL-37-resistance. Furthermore, a sublethal dosage of LL-37 stimulated the production of OMVs. Finally, we showed that OMVs produced by OmpT-positive strains protect the OmpT-negative strain, which is susceptible to LL-37 by itself more efficiently than OMVs from the ompT mutant. These results indicate that EHEC enhances the secretion of OmpT-loaded OMVs in coordination with the activation of virulence genes during infection and blocks bacterial cell attack by LL-37.
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