CD103+CD11b+ mucosal classical dendritic cells initiate long-term switched antibody responses to flagellin.

Antibody responses induced at mucosal and nonmucosal sites demonstrate a significant level of autonomy. Here, we demonstrate a key role for mucosal interferon regulatory factor-4 (IRF4)-dependent CD103+CD11b+ (DP), classical dendritic cells (cDCs) in the induction of T-dependent immunoglobulin G (IgG) and immunoglobulin A (IgA) responses in the mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103+CD11b- (SP) are not required for these responses. The lack of this response correlated with a complete absence of sFliC-specific plasma cells in the MLN, small intestinal lamina propria, and surprisingly also the bone marrow (BM). Many sFliC-specific plasma cells accumulating in the BM of immunized wild-type mice expressed α4ß7+, suggesting a mucosal origin. Collectively, these results suggest that mucosal DP cDC contribute to the generation of the sFliC-specific plasma cell pool in the BM and thus serve as a bridge linking the mucosal and systemic immune system.

C-terminal amino acid residues of the trimeric autotransporter adhesin YadA of Yersinia enterocolitica are decisive for its recognition and assembly by BamA.

The Bam complex is a highly conserved multiprotein machine essential for the assembly of ß-barrel outer membrane proteins. It is composed of the essential outer membrane protein BamA and four outer membrane associated lipoproteins BamB-E. The Yersinia enterocolitica Adhesin A (YadA) is the prototype of trimeric auotransporter adhesins (TAAs), consisting of a head, stalk and a ß-barrel membrane anchor. To investigate the role of BamA in biogenesis of TAAs, we expressed YadA in a BamA-depleted strain of Escherichia coli, which resulted in degradation of YadA. Yeast-two-hybrid experiments and immunofluorescence studies revealed that BamA and YadA interact directly and colocalize. As BamA recognizes the C-terminus of OMPs, we exchanged the nine most C-terminal amino acids of YadA. Substitution of the amino acids in position 1, 3 or 5 from the C-terminus with glycine resulted in DegP-dependent degradation of YadA. Despite degradation all YadA proteins assembled in the outer membrane. In summary we demonstrate that (i) BamA is essential for biogenesis of the TAA YadA, (ii) BamA interacts directly with YadA, (iii) the C-terminal amino acid motif of YadA is important for the BamA-dependent assembly and differs slightly compared with other OMPs, and (iv) BamA and YadA colocalize.

The orientation of a tandem POTRA domain pair, of the beta-barrel assembly protein BamA, determined by PELDOR spectroscopy.

The outer membrane beta-barrel trans-membrane proteins in gram-negative bacteria are folded into the membrane with the aid of polypeptide transport-associated (POTRA) domains. These domains occur, and probably function, as a tandem array situated on the periplasmic side of the outer membrane. Two crystal structures and one NMR study have attempted to define the structure and articulation of the POTRA domains of the Escherichia coli, prototypic Omp85 protein BamA. We have used pulsed electron paramagnetic resonance (EPR) to determine the distance and distance distribution between (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSSL), placed across the domain interface of the first two POTRA domains of BamA. Our results show tightly defined interdomain distance distributions that indicate a well-defined domain orientation. Examination of the known structures revealed that none of them fitted the EPR data. A combination of EPR and NMR data was used to generate converged structures with defined domain-domain orientation.

RNA processing and Arabidopsis flowering time control.

Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3'-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.

Polymorphic proteins of Chlamydia spp.--autotransporters beyond the Proteobacteria.

Gram-negative bacteria secrete a variety of proteins to the cell surface and beyond, a process with many inherent difficulties. An exceptionally widespread answer to these problems is the type V (or autotransporter) secretion pathway. By exploiting the data made available by bacterial genome sequencing, we have discovered that the previously described polymorphic proteins of Chlamydia spp. resemble members of the autotransporter family, and we suggest that they follow the same secretion pathway.

Enteroaggregative escherichia coli virulence factors in traveler's diarrhea strains.

Enteroaggregative Escherichia coli (EAEC) is associated with diarrhea in Spanish travelers to developing countries. In this study, the polymerase chain reaction was used to test EAEC isolates for genes encoding putative virulence factors, including EAEC adhesins, the plasmid-encoded toxin (Pet), a heat-stable enterotoxin (EAST), and Shigella enterotoxins 1 and 2 (ShET1 and ShET2). Findings included a low prevalence of genes for Pet (4.3%), ShET2 (4.3%), and the adherence factor AAF/II (8.7%). The overlapping genes encoding the ShET1 and the Pic mucinase were present in most EAEC strains tested (56.5%); however, some strains that carried this locus did not produce both proteins, as determined by Western immunoblot. Surprisingly, ShET1 and ShET2 genes were also found in other E. coli pathotypes, as was the EAST toxin locus. These findings underscore the heterogeneity of EAEC strains and suggest that the ShET1 may be an important virulence factor in traveler's diarrhea.

Identification of sat, an autotransporter toxin produced by uropathogenic Escherichia coli.

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.

The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation.

In this study, the sigA gene situated on the she pathogenicity island of Shigella flexneri 2a was cloned and characterized. Sequence analysis showed that sigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of Shigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.

Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli.

We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis.

Molecular switches--the ON and OFF of bacterial phase variation.

The expression of most bacterial genes is controlled at the level of transcription via promoter control mechanisms that permit a graded response. However, an increasing number of bacterial genes are found to exhibit an 'all-or-none' control mechanism that adapts the bacterium to more than one environment. One such mechanism is phase variation, traditionally defined as the high-frequency ON<-->OFF switching of phenotype expression. Phase variation events are usually random, but may be modulated by environmental conditions. The mechanisms of phase variation events and their significance within the microbial community are discussed here.

Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage.

Enteroaggregative Escherichia coli (EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity. We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins. When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa. Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture. We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein. Here, we have examined the effects of EAEC strain 042 and its isogenic pet mutant in the IVOC model. 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs. Colonic tissue incubated with the pet mutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter. Mucosal effects were restored upon complementation of the pet mutation in trans. Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet. The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage.

Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli.

The phylogenetics of the various pathotypes of diarrheagenic Escherichia coli are not completely understood. In this study, we identified several plasmid and chromosomal genes in the pathogenic enteroaggregative E. coli (EAEC) prototype strain 042 and determined the prevalence of these loci among EAEC and diffusely adherent E. coli strains. The distribution of these genes is analyzed within an evolutionary framework provided by the characterization of allelic variation in housekeeping genes via multilocus enzyme electrophoresis. Our data reveal that EAEC strains are heterogeneous with respect to chromosomal and plasmid-borne genes but that the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution.

The major phase-variable outer membrane protein of Escherichia coli structurally resembles the immunoglobulin A1 protease class of exported protein and is regulated by a novel mechanism involving Dam and oxyR.

Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). The agn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an alpha43 subunit (predicted Mr of 49,789) and a C-terminal domain (beta43) with features typical of a bacterial integral outer membrane protein (predicted Mr of 51, 642). Secondary structure analysis predicts that beta43 exists as an 18-stranded beta barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria and Haemophilus spp. The correct processing of the polyprotein to alpha43 and beta43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within alpha43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene.

Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli.

Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.

The great escape: structure and function of the autotransporter proteins.

The autotransporters, a family of secreted proteins from Gram-negative bacteria, possess an overall unifying structure comprising three functional domains: the amino-terminal leader sequence, the secreted mature protein (passenger domain) and a carboxy-terminal (beta-) domain that forms a beta-barrel pore to allow secretion of the passenger protein. Members of this family have been implicated as important or putative virulence factors in many Gram-negative pathogens.