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.

SummaryRapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage . Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor 32 . Proteolysis of 32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of 32 . Electron microscopy revealed that purified FtsH forms ringshaped structures with a diameter of 6-7 nm.

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.

ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein. Proteolysis by ATP-dependent proteases is an important mechanism for the rapid control of gene activity, the removal of unfolded inactive proteins, and the elimination of incomplete polypeptides. In bacteria, proteolysis acts on key regulatory transcription factors affecting the heat shock ( 32 ), stationary phase ( s ), and the SOS DNA repair system (LexA) responses, capsular polysaccharide biosynthesis, and the control of the lysis-lysogeny decision of phage (1). FtsH is a membranebound ATP-dependent protease in which the ATPase domain and the protease domain are linked in one polypeptide (2). FtsH orthologs are found within mitochondria and chloroplasts in higher organisms (3, 4). The number of native proteins known to be substrates for FtsH is rather small and includes the heat shock sigma factor 32 , SecY, YccA, subunit a of the membraneembedded F 0 part of the H ϩ -ATPase, as well as phage CII, CIII, and Xis proteins (see ref. 5). However, the signal(s) by which these native substrates are recognized by FtsH is not known. FtsH, as well as Tsp, ClpAP, and ClpXP recognize the SsrA peptide tag that is added to the C terminus of incomplete proteins by trans-translation (6).The lysogenic response established after infection of Escherichia coli by the temperate bacteriophage requires the initial synthesis of the CI repressor from the pE promoter and the integration protein Int, from the pI promoter. In addition, establishment of viable lysogenic cells requires the expression of the paQ promoter that inhibits lytic gene expression. The phage CII protein, which is 97 amino acid residues long, plays a key initiating role in these processes by activating the pE, pI, and paQ promoters (7). CII itself is regulated at many levels: transcription, translatio...

No abstract