The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein-injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10-kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung.

Nod1 and Nod2 are members of the Nod-like receptor family that detect intracellular bacterial peptidoglycan-derived muramyl peptides. The biological effects of muramyl peptides have been described for over three decades, but the mechanism underlying their internalization to the cytosol remains unclear. Using the human epithelial cell line HEK293T as a model system, we demonstrate here that Nod1-activating ligands entered cells through endocytosis, most likely by the clathrin-coated pit pathway, as internalization was dynamin-dependent but not inhibited by methyl-␤-cyclodextrin. In the endocytic pathway, the cytosolic internalization of Nod1 ligands was pH-dependent, occurred prior to the acidification mediated by the vacuolar ATPase, and was optimal at pH ranging from 5.5 to 6. Similarly, the Nod2 ligand MDP was internalized into host cytosol through a similar pathway with optimal pH for internalization ranging from 5.5 to 6.5. Moreover, Nod1-activating muramyl peptides likely required processing by endosomal enzymes, prior to transport into the cytosol, suggesting the existence of a sterically gated endosomal transporter for Nod1 ligands. In support for this, we identified a role for SLC15A4, an oligopeptide transporter expressed in early endosomes, in Nod1-dependent NF-B signaling. Interestingly, SLC15A4 expression was also up-regulated in colonic biopsies from patients with inflammatory bowel disease, a disorder associated with mutations in Nod1 and Nod2. Together, our results shed light on the mechanisms by which muramyl peptides get access to the host cytosol, where they are detected by Nod1 and Nod2, and might have implications for the understanding of human diseases, such as inflammatory bowel disease.

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.