Receptor chimeras demonstrate that the C-terminal domain of the human cytomegalovirus US27 gene product is necessary and sufficient for intracellular receptor localization

<p>Abstract</p> <p>Background</p> <p>Human cytomegalovirus (HCMV) is ubiquitous in the population but generally causes only mild or asymptomatic infection except in immune suppressed individuals. HCMV employs numerous strategies for manipulating infected cells, including mimicry of G-protein coupled receptors (GPCRs). The HCMV US27 gene product is a putative GPCR, yet no ligand or signaling has been identified for this receptor. In the present study, immunofluorescence microscopy was used to examine the cellular distribution of wild type US27, as well as US27 deletion mutants and chimeric receptors.</p> <p>Results</p> <p>In transiently transfected cells, wild type US27 was found primarily in intracellular compartments, in striking contrast to the cell surface distribution seen for the human cellular chemokine receptor CXCR3. When the N-terminal extracellular domains of the two receptors were swapped, no change in protein localization was observed. However, swapping of the C-terminal intracellular domains resulted in a significant change in receptor distribution. A chimera that contained US27 fused to the C-terminal intracellular tail of CXCR3 exhibited surface distribution similar to that of wild-type CXCR3. When the C-terminal domain of US27 was fused to CXCR3, this chimeric receptor (CXCR3/US27-CT) was found in the same intracellular pattern as wild-type US27. In addition, a US27 mutant lacking the C-terminus (US27ΔCT) failed to accumulate inside the cell and exhibited cell surface distribution. Co-localization with organelle-specific markers revealed that wild-type US27 was found predominantly in the Golgi apparatus and in endosomal compartments, whereas the US27/CXCR3-CT chimera, US27ΔCT and US27Δ348 mutants were not localized to endosomal compartments.</p> <p>Conclusions</p> <p>The results indicate that the C-terminal domain of the HCMV US27 protein, which contains a di-leucine endocytic sorting motif, is both necessary and sufficient for intracellular localization, which may also help explain why no cellular ligands have yet been identified for this viral receptor.</p

Diverse Intestinal Bacteria Contain Putative Zwitterionic Capsular Polysaccharides with Anti-inflammatory Properties

Zwitterionic capsular polysaccharides (ZPSs) are bacterial products that modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). However, only a few diverse bacteria are known to modulate the host immune system via ZPS. We present a genomic screen for bacteria encoding ZPS molecules. We identify diverse host-associated bacteria, including commensals and pathogens with known anti-inflammatory properties, with the capacity to produce ZPSs. Human mononuclear cells stimulated with lysates from putative ZPS-producing bacteria induce significantly greater IL-10 production and higher proportions of Tregs than lysates from non-ZPS-encoding relatives or a commensal strain of Bacteroides cellulosilyticus in which a putative ZPS biosynthetic operon was genetically disrupted. Similarly, wild-type B. cellulosilyticus DSM 14838, but not a close relative lacking a putative ZPS, attenuated experimental colitis in mice. Collectively, this screen identifies bacterial strains that may use ZPSs to interact with the host as well as those with potential probiotic properties

Diverse Intestinal Bacteria Contain Putative Zwitterionic Capsular Polysaccharides with Anti-inflammatory Properties

Zwitterionic capsular polysaccharides (ZPSs) are bacterial products that modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). However, only a few diverse bacteria are known to modulate the host immune system via ZPS. We present a genomic screen for bacteria encoding ZPS molecules. We identify diverse host-associated bacteria, including commensals and pathogens with known anti-inflammatory properties, with the capacity to produce ZPSs. Human mononuclear cells stimulated with lysates from putative ZPS-producing bacteria induce significantly greater IL-10 production and higher proportions of Tregs than lysates from non-ZPS-encoding relatives or a commensal strain of Bacteroides cellulosilyticus in which a putative ZPS biosynthetic operon was genetically disrupted. Similarly, wild-type B. cellulosilyticus DSM 14838, but not a close relative lacking a putative ZPS, attenuated experimental colitis in mice. Collectively, this screen identifies bacterial strains that may use ZPSs to interact with the host as well as those with potential probiotic properties