An E. coli display method for characterization of peptide-sensor kinase interactions.

Bacteria use two-component system (TCS) signaling pathways to sense and respond to peptides involved in host defense, quorum sensing and inter-bacterial warfare. However, little is known about the broad peptide-sensing capabilities of TCSs. In this study, we developed an Escherichia coli display method to characterize the effects of human antimicrobial peptides (AMPs) on the pathogenesis-regulating TCS PhoPQ of Salmonella Typhimurium with much higher throughput than previously possible. We found that PhoPQ senses AMPs with diverse sequences, structures and biological functions. We further combined thousands of displayed AMP variants with machine learning to identify peptide sub-domains and biophysical features linked to PhoPQ activation. Most of the newfound AMP activators induce PhoPQ in S. Typhimurium, suggesting possible roles in virulence regulation. Finally, we present evidence that PhoPQ peptide-sensing specificity has evolved across commensal and pathogenic bacteria. Our method enables new insights into the specificities, mechanisms and evolutionary dynamics of TCS-mediated peptide sensing in bacteria.

TGFß regulates Galectin-3 expression through canonical Smad3 signaling pathway in nucleus pulposus cells: implications in intervertebral disc degeneration.

Galectin-3 is highly expressed in notochordal nucleus pulposus (NP) and thought to play important physiological roles; however, regulation of its expression remains largely unexplored. The aim of the study was to investigate if TGFß regulates Galectin-3 expression in NP cells. TGFß treatment resulted in decreased Galectin-3 expression. Bioinformatic analysis using JASPAR and MatInspector databases cross-referenced with published ChIP-Seq data showed nine locations of highly probable Smad3 binding in the LGALS3 proximal promoter. In NP cells, TGFß treatment resulted in decreased activity of reporters harboring several 5' deletions of the proximal Galectin-3 promoter. While transfection of NP cells with constitutively active (CA)-ALK5 resulted in decreased promoter activity, DN-ALK5 blocked the suppressive effect of TGFß on the promoter. The suppressive effect of Smad3 on the Galectin-3 promoter was confirmed using gain- and loss-of-function studies. Transfection with DN-Smad3 or Smad7 blocked TGFß mediated suppression of promoter activity. We also measured Galectin-3 promoter activity in Smad3 null and wild type cells. Noteworthy, promoter activity was suppressed by TGFß only in wild type cells. Likewise, stable silencing of Smad3 in NP cells using sh-Smad3 significantly blocked TGFß-dependent decrease in Galectin-3 expression. Treatment of human NP cells isolated from tissues with different grades of degeneration showed that Galectin-3 expression was responsive to TGF-ß-mediated suppression. Importantly, Galectin-3 synergized effects of TNF-α on inflammatory gene expression by NP cells. Together these studies suggest that TGFß, through Smad3 controls Galectin-3 expression in NP cells and may have implications in the intervertebral disc degeneration.