Chlamydia trachomatis antigens in enteroendocrine cells and macrophages of the small bowel in patients with severe irritable bowel syndrome

<p>Abstract</p> <p>Background</p> <p>Inflammation and immune activation have repeatedly been suggested as pathogentic factors in irritable bowel syndrome (IBS). The driving force for immune activation in IBS remains unknown. The aim of our study was to find out if the obligate intracellular pathogen <it>Chlamydia </it>could be involved in the pathogenesis of IBS.</p> <p>Methods</p> <p>We studied 65 patients (61 females) with IBS and 42 (29 females) healthy controls in which IBS had been excluded. Full thickness biopsies from the jejunum and mucosa biopsies from the duodenum and the jejunum were stained with a monoclonal antibody to <it>Chlamydia </it>lipopolysaccharide (LPS) and species-specific monoclonal antibodies to <it>C. trachomatis </it>and <it>C. pneumoniae</it>. We used polyclonal antibodies to chromogranin A, CD68, CD11c, and CD117 to identify enteroendocrine cells, macrophages, dendritic, and mast cells, respectively.</p> <p>Results</p> <p><it>Chlamydia </it>LPS was present in 89% of patients with IBS, but in only 14% of healthy controls (p < 0.001) and 79% of LPS-positive biopsies were also positive for <it>C. trachomatis </it>major outer membrane protein (MOMP). Staining for <it>C. pneumoniae </it>was negative in both patients and controls. <it>Chlamydia </it>LPS was detected in enteroendocrine cells of the mucosa in 90% of positive biopsies and in subepithelial macrophages in 69% of biopsies. Biopsies taken at different time points in 19 patients revealed persistence of <it>Chlamydia </it>LPS up to 11 years. The odds ratio for the association of <it>Chlamydia </it>LPS with presence of IBS (43.1; 95% CI: 13.2-140.7) is much higher than any previously described pathogenetic marker in IBS.</p> <p>Conclusions</p> <p>We found <it>C. trachomatis </it>antigens in enteroendocrine cells and macrophages in the small bowel mucosa of patients with IBS. Further studies are required to clarify if the presence of such antigens has a role in the pathogenesis of IBS.</p

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics

Accurate Prediction of Secreted Substrates and Identification of a Conserved Putative Secretion Signal for Type III Secretion Systems

The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates—effector proteins—are not. We have used a novel computational approach to confidently identify new secreted effectors by integrating protein sequence-based features, including evolutionary measures such as the pattern of homologs in a range of other organisms, G+C content, amino acid composition, and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from the plant pathogen Pseudomonas syringae and validated on a set of effectors from the animal pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) after eliminating effectors with detectable sequence similarity. We show that this approach can predict known secreted effectors with high specificity and sensitivity. Furthermore, by considering a large set of effectors from multiple organisms, we computationally identify a common putative secretion signal in the N-terminal 20 residues of secreted effectors. This signal can be used to discriminate 46 out of 68 total known effectors from both organisms, suggesting that it is a real, shared signal applicable to many type III secreted effectors. We use the method to make novel predictions of secreted effectors in S. Typhimurium, some of which have been experimentally validated. We also apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis, identifying the majority of known secreted proteins in addition to providing a number of novel predictions. This approach provides a new way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal