Ben J. Appelmelk

Mycobacteria Target DC-SIGN to Suppress Dendritic Cell Function

Mycobacterium tuberculosis represents a world-wide health risk and immunosuppression is a particular problem in M. tuberculosis infections. Although macrophages are primarily infected, dendritic cells (DCs) are important in inducing cellular immune responses against M. tuberculosis. We hypothesized that DCs represent a target for M. tuberculosis and that the observed immuno-suppression results from modulation of DC functions. We demonstrate that the DC-specific C-type lectin DC-SIGN is an important receptor on DCs that captures and internalizes intact Mycobacterium bovis bacillus Calmette-Guérin (BCG) through the mycobacterial cell wall component ManLAM. Antibodies against DC-SIGN block M. bovis BCG infection of DCs. ManLAM is also secreted by M. tuberculosis–infected macrophages and has been implicated as a virulence factor. Strikingly, ManLAM binding to DC-SIGN prevents mycobacteria- or LPS-induced DC maturation. Both mycobacteria and LPS induce DC maturation through Toll-like receptor (TLR) signaling, suggesting that DC-SIGN, upon binding of ManLAM, interferes with TLR-mediated signals. Blocking antibodies against DC-SIGN reverse the ManLAM-mediated immunosuppressive effects. Our results suggest that M. tuberculosis targets DC-SIGN both to infect DCs and to down-regulate DC-mediated immune responses. Moreover, we demonstrate that DC-SIGN has a broader pathogen recognition profile than previously shown, suggesting that DC-SIGN may represent a molecular target for clinical intervention in infections other than HIV-1.

Type VII secretion — mycobacteria show the way

Recent evidence shows that mycobacteria have developed novel and specialized secretion systems for the transport of extracellular proteins across their hydrophobic, and highly impermeable, cell wall. Strikingly, mycobacterial genomes encode up to five of these transport systems. Two of these systems, ESX-1 and ESX-5, are involved in virulence — they both affect the cell-to-cell migration of pathogenic mycobacteria. Here, we discuss this novel secretion pathway and consider variants that are present in various Gram-positive bacteria. Given the unique composition of this secretion system, and its general importance, we propose that, in line with the accepted nomenclature, it should be called type VII secretion.

Cutting Edge: Carbohydrate Profiling Identifies New Pathogens That Interact with Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin on Dendritic Cells

Dendritic cells (DC) are instrumental in handling pathogens for processing and presentation to T cells, thus eliciting an appropriate immune response. C-type lectins expressed by DC function as pathogen-recognition receptors; yet their specificity for carbohydrate structures on pathogens is not fully understood. In this study, we analyzed the carbohydrate specificity of DC-specific ICAM-3-grabbing nonintegrin (SIGN)/CD209, the recently documented HIV-1 receptor on DC. Our studies show that DC-SIGN binds with high affinity to both synthetic mannose- and fucose-containing glycoconjugates. These carbohydrate structures are abundantly expressed by pathogens as demonstrated by the affinity of DC-SIGN for natural surface glycans of the human pathogens Mycobacterium tuberculosis, Helicobacter pylori, Leishmania mexicana, and Schistosoma mansoni. This analysis expands our knowledge on the carbohydrate and pathogen-specificity of DC-SIGN and identifies this lectin to be central in pathogen-DC interactions.

Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN

The human gastric pathogen Helicobacter pylori spontaneously switches lipopolysaccharide (LPS) Lewis (Le) antigens on and off (phase-variable expression), but the biological significance of this is unclear. Here, we report that Le+ H. pylori variants are able to bind to the C-type lectin DC-SIGN and present on gastric dendritic cells (DCs), and demonstrate that this interaction blocks T helper cell (Th)1 development. In contrast, Le− variants escape binding to DCs and induce a strong Th1 cell response. In addition, in gastric biopsies challenged ex vivo with Le+ variants that bind DC-SIGN, interleukin 6 production is decreased, indicative of increased immune suppression. Our data indicate a role for LPS phase variation and Le antigen expression by H. pylori in suppressing immune responses through DC-SIGN.

Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28 h‐old zebrafish embryos were infected with DsRed‐labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild‐type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage‐like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non‐pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage‐like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat‐killed, wild‐type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non‐invasive and real time analysis of bacterial infections in a vertebrate host.

The gastric H+,K+-ATPase is a major autoantigen in chronic Helicobacter pylori gastritis with body mucosa atrophy ☆ ☆☆ ★ ★★

BACKGROUND & AIMS A subgroup of Helicobacter pylori-infected patients develops autoantibodies to gastric parietal cell canaliculi. The aim of this study was to define the unknown autoantigen. METHODS We screened 72 H. pylori-infected patients, 5 patients with autoimmune gastritis, and 36 healthy controls for immunoglobulin G autoantibodies to canaliculi by immunohistochemistry. The antigen specificity was determined by immunoprecipitation of the murine gastric H+,K+-adenosine triphosphatase (H+,K+-ATPase) expressed in oocytes and by immunoblotting on human gastric membranes from the body mucosa. RESULTS Autoantibodies specific for the conformational peptides of the H+,K+-ATPase were detected in 3% (1/36) of controls, in all patients with autoimmune gastritis (5/5), in 25% (18/72) of H. pylori-infected patients, and in 47% (15/32) of the infected patients with anticanalicular autoantibodies. No other major autoantigen was identified. Atrophy in the gastric body mucosa was found in 60% (9/15) of infected patients with both anticanalicular and anti-H+,K+-ATPase antibodies, but only in 13% (5/37) of infected patients lacking both autoantibodies (P < 0.01). CONCLUSIONS The gastric H+,K+-ATPase is a major autoantigen in H. pylori-associated antigastric autoimmunity. Thus, anti-H+,K+-ATPase autoantibodies, which are closely linked to classical autoimmune gastritis, are also significant indicators for body mucosa atrophy in chronic H. pylori gastritis.

Molecular Mimicry between Helicobacter pylori Antigens and H+,K+–Adenosine Triphosphatase in Human Gastric Autoimmunity

Autoimmune gastritis and Helicobacter pylori–associated gastric atrophy develop through similar mechanisms involving the proton pump H+,K+–adenosine triphosphatase as autoantigen. Here, we report that H. pylori–infected patients with gastric autoimmunity harbor in vivo–activated gastric CD4+ T cells that recognize both H+,K+–adenosine triphosphatase and H. pylori antigens. We characterized the submolecular specificity of such gastric T cells and identified cross-reactive epitopes from nine H. pylori proteins. Cross-reactive H. pylori peptides induced T cell proliferation and expression of T helper type 1 functions. We suggest that in genetically susceptible individuals, H. pylori infection can activate cross-reactive gastric T cells leading to gastric autoimmunity via molecular mimicry.

A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.

Mycobacterial genomes contain two unique gene families, the so‐called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. As PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT‐6‐like secretion system. This cluster, designated ESX‐5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M. smegmatis. Only introduction of the complete ESX‐5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, as the ESX‐5 mutant of M. marinum was affected in spreading to uninfected macrophages.

Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host–pathogen interaction

Approximately one third of the worlds population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl–arabinogalactan–peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host–pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host–pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates.

Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.