Two major arms of the inflammatory response are the NF-jB and c-Jun N-terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn-dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF-jB and AP-1 activation. We show that NleC and NleD co-operate and complement other EPEC effectors in accomplishing maximal inhibition of IL-8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.

The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-κB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-κB activation. We show that inhibition of NF-κB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-κB activation by preventing activation of IKKβ and consequently the degradation of the NF-κB inhibitor, IκB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-κB signaling pathways.

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes.Colonizing enteropathogens compete with the gut flora to gain a foothold in the host tissue by expressing powerful colonization factors. However, to reduce the immune response of the host, the pathogen should minimize the expression of the colonization factors. To resolve this dilemma, pathogens evolved regulatory mechanisms that optimize the expression levels and timing, thus maintaining expression of just enough colonization factors and only when needed. Another layer of complexity is added when the colonization is dependent on the assembly of organelles like the type III secretion systems (TTSS), which are composed of ϳ30 different proteins of various relative amounts and encoded by several operons. In these cases, an orderly expression program is required for efficient assembly of the organelle.Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. It employs the TTSS as a molecular syringe to inject a battery of toxic or colonization proteins into the membrane and cytoplasm of infected host cells (4). The TTSS and some of the effectors are encoded by a 35.6-kbp pathogenicity island, termed the locus for enterocyte effacement (LEE). The LEE consists of 41 genes, organized in five major operons (LEE1 to LEE5) and several additional transcriptional units (10,19). Ler, an H-NS paralog, encoded by the first gene of the LEE1 operon, is a key regulator of the LEE regulon, positively regulating expression of LEE2, LEE3, LEE4, LEE5, espG, and map (11,19,24,30). The regulation of ler (LEE1 operon) is complex and involves many factors, including H-NS, integration host factor (IHF), Fis, PerC, BipA, GrlA, GrlR, GadX, and quorum sensing (2,7,11,13,14,17,19,26,28,30,32). Most of these factors appe...

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