Combined multidimensional single-cell protein and RNA profiling dissects the cellular and functional heterogeneity of thymic epithelial cells.

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T cell development and selection. Recent single-cell RNA sequencing studies have uncovered previously unappreciated transcriptional heterogeneity among thymic epithelial cells (TEC). However, there are only very few cell markers that allow a comparable phenotypic identification of TEC.

Developmental dynamics of the neural crest-mesenchymal axis in creating the thymic microenvironment.

The thymic stroma is composed of epithelial and nonepithelial cells providing separate microenvironments controlling homing, differentiation, and selection of hematopoietic precursor cells to functional T cells. Here, we explore at single-cell resolution the complex composition and dynamic changes of the nonepithelial stromal compartment across different developmental stages in the human and mouse thymus, and in an experimental model of the DiGeorge syndrome, the most common form of human thymic hypoplasia. The detected gene expression signatures identify previously unknown stromal subtypes and relate their individual molecular profiles to separate differentiation trajectories and functions, revealing an unprecedented heterogeneity of different cell types that emerge at discrete developmental stages and vary in their expression of key regulatory signaling circuits and extracellular matrix components.

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function.

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired.

Ageing compromises mouse thymus function and remodels epithelial cell differentiation.

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear.

Keratinocyte growth factor impairs human thymic recovery from lymphopenia.

Background: The lymphocyte-depleting antibody alemtuzumab is a highly effective treatment of relapsing-remitting multiple sclerosis (RRMS); however 50% of patients develop novel autoimmunity post-treatment. Most at risk are individuals who reconstitute their T-cell pool by proliferating residual cells, rather than producing new T-cells in the thymus; raising the possibility that autoimmunity might be prevented by increasing thymopoiesis. Keratinocyte growth factor (palifermin) promotes thymopoiesis in non-human primates.

Crystal Structure of a Complex of Surfactant Protein D (SP-D) and Haemophilus influenzae Lipopolysaccharide Reveals Shielding of Core Structures in SP-D-Resistant Strains.

The carbohydrate recognition domains (CRDs) of lung collectin surfactant protein D (SP-D) recognize sugar patterns on the surface of lung pathogens and promote phagocytosis. Using Haemophilus influenzae Eagan strains expressing well-characterized lipopolysaccharide (LPS) surface structures of various levels of complexity, we show that bacterial recognition and binding by SP-D is inversely related to LPS chain extent and complexity. The crystal structure of a biologically active recombinant trimeric SP-D CRD complexed with a delipidated Eagan 4A LPS suggests that efficient LPS recognition by SP-D requires multiple binding interactions utilizing the three major ligand-binding determinants in the SP-D binding pocket, with Ca-dependent binding of inner-core heptose accompanied by interaction of anhydro-Kdo (4,7-anhydro-3-deoxy-d-manno-oct-2-ulosonic acid) with Arg343 and Asp325.

Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia.

Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire).

Construction of Opa-positive and Opa-negative strains of Neisseria meningitidis to evaluate a novel meningococcal vaccine.

Neisseria meningitidis is a major global pathogen causing invasive disease with a mortality of 5-10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opacity-associated adhesin (Opa) proteins are major meningococcal outer membrane proteins, which have shown recent promise as a potential novel vaccine.

Detailed structural features of lipopolysaccharide glycoforms in nontypeable Haemophilus influenzae strain 2019.

We have investigated the structure of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 2019, a prototype strain that is used for studies of NTHi biology and disease. Analysis of LPS from wild type and lex2B, lpt3 and pgm mutant strains using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MS(n) on permethylated dephosphorylated OS, confirmed the previously established structure in which lactose is linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-α-D-Hepp-(1→2)-[PEtn→6]-l-α-D-Hepp-(1→3)-l-α-D-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. Importantly, our data provide further structural detail whereby extensions from the middle heptose (HepII) are now characterized as β-D-Galp-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→3 and truncated versions thereof.

The role of lic2B in lipopolysaccharide biosynthesis in Haemophilus influenzae strain Eagan.

Lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae involves genes from the lic2 locus that are required for chain extension from the middle heptose (HepII) of the conserved triheptosyl inner-core moiety. Lic2C initiates the process by attaching the first glucose to HepII, but the gene encoding for the enzyme adding the next β-D-Glcp- is uncharacterized. Lic2B is the candidate glucosyltransferase; however, in previous investigations, mutation of lic2B resulted in no hexose extension from HepII, likely due to a polar effect on the lic2C gene.

Genes required for the synthesis of heptose-containing oligosaccharide outer core extensions in Haemophilus influenzae lipopolysaccharide.

Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either l-glycero-d-manno-heptose (ld-Hep) or d-glycero-d-manno-heptose (dd-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases.

Transposon insertion in a serine-specific minor tRNA coding sequence affects intraperitoneal survival of Haemophilus influenzae in the infant rat model.

Due to its lifestyle as a commensal and occasional pathogen in the upper and lower respiratory tracts of humans, Haemophilus influenzae needs to protect itself from endogenously and exogenously generated reactive oxygen species. To better understand the oxygen radical resistance and to investigate a correlation with virulence, randomly generated paraquat-sensitive H. influenzae transposon mutants were analyzed in an infant rat model of infection.

A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019.

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-GlcIp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-alpha-Kdop) to which addition of beta-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a beta-d-Galp is linked to O-4 of GlcI.

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain R2846.

We here report the lipopolysaccharide (LPS) structures expressed by nontypeable Haemophilus influenzae R2846, a strain whose complete genome sequence has recently been obtained. Results were obtained by using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS (n) on permethylated dephosphorylated OS. A beta- d-Glc p-(1-->4)- d-alpha- d-Hep p-(1-->6)-beta- d-Glc p-(1-->4) unit was found linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-alpha- d-Hep p-(1-->2)-[ PEtn-->6]- l-alpha- d-Hep p-(1-->3)- l-alpha- d-Hep p-(1-->5)-[ PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A.

Haemophilus influenzae biofilms: hypothesis or fact?

Many publications state that nontypeable Haemophilus influenzae (NTHi) produces biofilms. Here, we review many of the publications that have led to acceptance by some that NTHi expresses a biofilm-specific phenotype as a distinct part of its life cycle. Biofilm formation was originally invoked to explain the failure to culture NTHi from middle-ear effusions, recalcitrance to antibiotics and its pathogenic behaviour.

Specific amino acids of the glycosyltransferase LpsA direct the addition of glucose or galactose to the terminal inner core heptose of Haemophilus influenzae lipopolysaccharide via alternative linkages.

Lipopolysaccharide is the major glycolipid of the cell wall of the bacterium Haemophilus influenzae, a Gram-negative commensal and pathogen of humans. Lipopolysaccharide is both a virulence determinant and a target for host immune responses. Glycosyltransferases have high donor and acceptor substrate specificities that are generally limited to catalysis of one unique glycosidic linkage.

Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O-acetyl groups.

Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes.

Three genes, lgtF, lic2C and lpsA, have a primary role in determining the pattern of oligosaccharide extension from the inner core of Haemophilus influenzae LPS.

Lipopolysaccharide (LPS) is a virulence determinant of Haemophilus influenzae and exhibits substantial heterogeneity in structure within and between strains. Key factors contributing to this heterogeneity are the genes required to add the first glycose to each of the three heptose residues of the LPS inner core. In each case this addition can facilitate further oligosaccharide extension.

High rates of recombination in otitis media isolates of non-typeable Haemophilus influenzae.

Non-typeable (NT) or capsule-deficient, Haemophilus influenzae (Hi) is a common commensal of the upper respiratory tract of humans and can be pathogenic resulting in diseases such as otitis media, sinusitis and pneumonia. The lipopolysaccharide (LPS) of NTHi is a major virulence factor that displays substantial intra-strain and inter-strain variation of its oligosaccharide structures. To investigate the genetic basis of LPS variation we sequenced internal regions of each of seven genes required for the biosynthesis of either the inner or the outer core oligosaccharide structures.

The avian chorioallantoic membrane in ovo--a useful model for bacterial invasion assays.

The aim of this study was to evaluate the practicability of the chick embryo chorioallantoic membrane (CAM) with special regard to the 'natural air sac' technique (NAST) of preparation for in-vivo research on the invasive potential of bacterial strains of various enterobacterial species. It was sought to establish an experimental system more closely resembling in-vivo conditions than cell lines on one hand, and cheaper and easier to handle than established animal models on the other. Fertilized eggs of the domestic fowl were incubated.

Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae.

A sialylated lacto-N-neotetraose (Sial-lNnT) structural unit was identified and structurally characterized in the lipopolysaccharide (LPS) from the genome-sequenced strain Rd [corrected] (RM118) of the human pathogen Haemophilus influenzae grown in the presence of sialic acid. A combination of molecular genetics, MS and NMR spectroscopy techniques showed that this structural unit extended from the proximal heptose residue of the inner core region of the LPS molecule. The structure of the Sial-lNnT unit was identical to that found in meningococcal LPS, but glycoforms containing truncations of the Sial-lNnT unit, comprising fewer residues than the complete oligosaccharide component, were not detected.

An in silico evaluation of Tn916 as a tool for generalized mutagenesis in Haemophilus influenzae Rd.

The transposon Tn916 was evaluated as a tool for generalized mutagenesis of the genome of Haemophilus influenzae. This was achieved in silico by searching the genome sequence of H. influenzae Rd for the published Tn916 target site consensus sequence 5' TT/ATTTT(N)6AAAAAA/TA.