CRISPR-based functional genomics in human dendritic cells.
Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.
View details for DOI 10.7554/eLife.65856
View details for PubMedID 33904395
The Biosynthesis of Lipooligosaccharide fromBacteroides thetaiotaomicron.
2018; 9 (2)
Lipopolysaccharide (LPS), a cell-associated glycolipid that makes up the outer leaflet of the outer membrane of Gram-negative bacteria, is a canonical mediator of microbe-host interactions. The most prevalent Gram-negative gut bacterial taxon,Bacteroides, makes up around 50% of the cells in a typical Western gut; these cells harbor ~300 mg of LPS, making it one of the highest-abundance molecules in the intestine. As a starting point for understanding the biological function ofBacteroidesLPS, we have identified genes inBacteroides thetaiotaomicronVPI 5482 involved in the biosynthesis of its lipid A core and glycan, generated mutants that elaborate altered forms of LPS, and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to interrogate the molecular features of these variants. We demonstrate,inter alia, that the glycan does not appear to have a repeating unit, and so this strain produces lipooligosaccharide (LOS) rather than LPS. This result contrasts withBacteroides vulgatusATCC 8482, which by SDS-PAGE analysis appears to produce LPS with a repeating unit. Additionally, our identification of theB. thetaiotaomicronLOS oligosaccharide gene cluster allowed us to identify similar clusters in otherBacteroidesspecies. Our work lays the foundation for developing a structure-function relationship forBacteroidesLPS/LOS in the context of host colonization.IMPORTANCEMuch is known about the bacterial species and genes that make up the human microbiome, but remarkably little is known about the molecular mechanisms through which the microbiota influences host biology. A well-known mechanism by which bacteria influence the host centers around lipopolysaccharide (LPS), a component of the Gram-negative bacterial outer membrane. Pathogen-derived LPS is a potent ligand for host receptor Toll-like receptor 4, which plays an important role in sensing bacteria as part of the innate immune response. Puzzlingly, the most common genus of human gut bacteria,Bacteroides, produces LPS but does not elicit a potent proinflammatory response. Previous work showing thatBacteroidesLPS differs structurally from pathogen-derived LPS suggested the outlines of an explanation. Here, we take the next step, elucidating the biosynthetic pathway forBacteroidesLPS and generating mutants in the process that will be of great use in understanding how this molecule modulates the host immune response.
View details for DOI 10.1128/mBio.02289-17
View details for PubMedID 29535205
Mammalian Lipopolysaccharide Receptors Incorporated into the Retroviral Envelope Augment Virus Transmission
CELL HOST & MICROBE
2015; 18 (4): 456-462
The orally transmitted retrovirus mouse mammary tumor virus (MMTV) requires the intestinal microbiota for persistence. Virion-associated lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), stimulating production of the immunosuppressive cytokine IL-10 and MMTV evasion of host immunity. However, the mechanisms by which MMTV associates with LPS remain unknown. We find that the viral envelope contains the mammalian LPS-binding factors CD14, TLR4, and MD-2, which, in conjunction with LPS-binding protein (LBP), bind LPS to the virus and augment transmission. MMTV isolated from infected mice lacking these LBPs cannot engage LPS or stimulate TLR4 and have a transmission defect. Furthermore, MMTV incorporation of a weak agonist LPS from Bacteroides, a prevalent LPS source in the gut, significantly enhances the ability of this LPS to stimulate TLR4, suggesting that MMTV intensifies these immunostimulatory properties. Thus, an orally transmitted retrovirus can capture, modify, and exploit mammalian receptors for bacterial ligands to ensure successful transmission.
View details for DOI 10.1016/j.chom.2015.09.005
View details for Web of Science ID 000365111600013
View details for PubMedID 26468748
View details for PubMedCentralID PMC4795803
A Phase-Variable Surface Layer from the Gut Symbiont Bacteroides thetaiotaomicron
2015; 6 (5)
The capsule from Bacteroides, a common gut symbiont, has long been a model system for studying the molecular mechanisms of host-symbiont interactions. The Bacteroides capsule is thought to consist of an array of phase-variable polysaccharides that give rise to subpopulations with distinct cell surface structures. Here, we report the serendipitous discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron: a surface layer composed of a protein of unknown function, BT1927. BT1927, which is expressed in a phase-variable manner by ~1:1,000 cells in a wild-type culture, forms a hexagonally tessellated surface layer. The BT1927-expressing subpopulation is profoundly resistant to complement-mediated killing, due in part to the BT1927-mediated blockade of C3b deposition. Our results show that the Bacteroides surface structure is capable of a far greater degree of structural variation than previously known, and they suggest that structural variation within a Bacteroides species is important for productive gut colonization.Many bacterial species elaborate a capsule, a structure that resides outside the cell wall and mediates microbe-microbe and microbe-host interactions. Species of Bacteroides, the most abundant genus in the human gut, produce a capsule that consists of an array of polysaccharides, some of which are known to mediate interactions with the host immune system. Here, we report the discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron. We show that this protein-based structure is expressed by a subset of cells in a population and protects Bacteroides from killing by complement, a component of the innate immune system. This novel surface layer protein is conserved across many species of the genus Bacteroides, suggesting an important role in colonization and host immune modulation.
View details for DOI 10.1128/mBio.01339-15
View details for Web of Science ID 000364523100038
View details for PubMedID 26419879
View details for PubMedCentralID PMC4611039
- Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers. PloS one 2015; 10 (10)