Monocyte exposure to LPS induces a transient state in which these cells are refractory to further endotoxin stimulation. This phenomenon, termed endotoxin tolerance (ET), is characterized by a decreased production of cytokines in response to the proinflammatory stimulus. We have established a robust model of ET and have determined the time frame and features of LPS unresponsiveness in cultured human monocytes. A large number of genes transcribed in tolerant monocytes were classified as either "tolerizable" or "nontolerizable" depending on their expression levels during the ET phase. Tolerant monocytes exhibit rapid IL-1R-associated kinase-M (IRAK-M) overexpression, high levels of triggering receptor expressed on myeloid cells-1 (TREM-1) and CD64, and a marked down-regulation of MHC molecules and NF-B2. These cells combine potent phagocytic activity with impaired capability for Ag presentation. We also show that circulating monocytes isolated from cystic fibrosis patients share all the determinants that characterize cells locked in an ET state. These findings identify a new mechanism that contributes to impaired inflammation in cystic fibrosis patients despite a high frequency of infections. Our results indicate that a tolerant phenotype interferes with timing, efficiency, and outcome of the innate immune responses against bacterial infections.

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

Intimin is a bacterial adhesin located on the surface of enteropathogenic Escherichia coli and other related bacteria that is believed to self-translocate across the outer membrane (OM), and therefore it has been regarded as a member of the type V secretion system (T5SS), which includes classical autotransporters (ATs). However, intimin has few structural similarities to classical ATs and an opposite topology with an OMembedded N region and a secreted C region. Since the actual secretion mechanism of intimin is unknown, we investigated intimin biogenesis by analyzing its requirement of periplasmic chaperones (DsbA, SurA, Skp, and DegP) and of OM protein BamA (YaeT/Omp85) for folding, OM insertion, and translocation. Using full-length and truncated intimin polypeptides, we demonstrate that DsbA catalyzes the formation of a disulfide bond in the D3 lectin-like domain of intimin in the periplasm, indicating that this secreted C-terminal domain is at least partially folded prior to its translocation across the OM. We also show that SurA chaperone plays the major role for periplasmic transport and folding of the N region of intimin, whereas the parallel pathway made by Skp and DegP chaperones plays a secondary role in this process. Further, we demonstrate that BamA is essential for the insertion of the N region of intimin in the OM and that the protease activity of DegP participates in the degradation of misfolded intimin. The significance of these findings for a BamA-dependent secretion mechanism of intimin is discussed in the context of T5SSs.Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) strains are important pathogens causing severe diarrheal diseases and other acute symptoms (e.g., hemorrhagic colitis and hemolytic-uremic syndrome) (31). EPEC and EHEC bacteria adhere to the intestinal enterocytes, inducing a destruction of microvilli called attaching and effacing (A/E) lesions, which involves a strong rearrangement of host cell cytoskeleton and the formation of an actin "pedestal" beneath the adherent bacteria. The formation of A/E lesions requires an intimate attachment of the bacterium to the host cell membrane mediated by the interaction of a specific bacterial adhesin, intimin (eaeA), with its receptor in the host cell membrane, Tir (for translocated intimin receptor), which is translocated by the bacterium using a type III secretion system (19). Most genes required for A/E lesion formation (i.e., eaeA, tir, and those encoding the type III secretion system) are clustered in a pathogenicity island referred to as the locus of enterocyte effacement (LEE) (16). The LEE is also found in the genome of other intestinal pathogens that induce similar A/E lesions, such as Citrobacter rodentium (13).Intimins are large polypeptides (ca. 95 kDa) displayed at the surfaces of LEE ϩ pathogenic bacteria that contain distinct structural and functional N and C regions (Fig. 1A) (26). The N region of intimins (residues 1 to 550) anchors the protein in the bacterial envelope and is highly c...