The Cytolethal Distending Toxin Subunit CdtB of Helicobacter Induces a Th17-related and Antimicrobial Signature in Intestinal and Hepatic Cells In Vitro.

Enterohepatic Helicobacter species are associated with several digestive diseases. Helicobacter pullorum is an emerging human foodborne pathogen, and Helicobacter hepaticus is a mouse pathogen; both species are associated with intestinal and/or hepatic diseases. They possess virulence factors, such as cytolethal distending toxin (CDT). Data indicate that CDT may be involved in chronic inflammatory responses, via its active subunit, CdtB. The proinflammatory properties of the CdtB of H. pullorum and H. hepaticus were assessed on human intestinal and hepatic epithelial cells in vitro. Interleukin 8 expression was evaluated by using wild-type strains and their corresponding CdtB isogenic mutants and by delivering CdtB directly into the cells. Nuclear factor κB nuclear translocation and transcriptomic characteristics in response to CdtB were also evaluated. The CdtB of these Helicobacter species induced nuclear factor κB nuclear translocation and exhibited proinflammatory properties, mainly the expression of T-helper type 17-related genes and genes encoding antimicrobial products also involved in cancer. The Histidine residue in position 265 of the CdtB catalytic site appeared to play a role in the regulation of most of these genes. As for flagellin or lipopolysaccharides, CdtB also induced expression of inflammation-associated genes related to antimicrobial activity.

Helicobacter pullorum cytolethal distending toxin targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial cells.

Helicobacter pullorum, a bacterium initially isolated from poultry, has been associated with human digestive disorders. However, the factor responsible for its cytopathogenic effects on epithelial cells has not been formally identified. The cytopathogenic alterations induced by several human and avian H. pullorum strains were investigated on human intestinal epithelial cell lines. Moreover, the effects of the cytolethal distending toxin (CDT) were evaluated first by using a wild-type strain and its corresponding cdtB isogenic mutant and second by delivering the active CdtB subunit of the CDT directly into the cells. All of the H. pullorum strains induced cellular distending phenotype, actin cytoskeleton remodeling, and G2/M cell cycle arrest. These effects were dependent on the CDT, as they were (1) not observed in response to a cdtB isogenic mutant strain and (2) present in cells expressing CdtB. CdtB also induced an atypical delocalization of vinculin from focal adhesions to the perinuclear region, formation of cortical actin-rich large lamellipodia with an upregulation of cortactin, and decreased cellular adherence. In conclusion, the CDT of H. pullorum is responsible for major cytopathogenic effects in vitro, confirming its role as a main virulence factor of this emerging human pathogen.

Control of Plasmodium falciparum erythrocytic cycle: γδ T cells target the red blood cell-invasive merozoites.

The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human Vγ9Vδ2 γδ T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate Vγ9Vδ2 T cells in a γδ T cell receptor-dependent manner and trigger their degranulation. In contrast, the γδ T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing Vδ2(+) T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of Vγ9Vδ2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies Vγ9Vδ2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of Vγ9Vδ2 T cells to control parasite density in malaria patients.

Activation of the receptor NKG2D leads to production of Th17 cytokines in CD4+ T cells of patients with Crohn's disease.

BACKGROUND & AIMS: The natural killer group 2 member D (NKG2D) is a stimulatory receptor expressed on a subset of mucosal and peripheral CD4+ T cells in patients with Crohn's disease (CD) and other inflammatory diseases. Ligand activation of NKG2D in patients induces CD4+ T cells to release T-helper (Th) 1 cytokines and become cytotoxic. We investigated the Th17 cytokines produced by T cells that express NKG2D in blood and intestinal mucosa samples from patients with CD. METHODS: We isolated CD4+ T cells from peripheral blood and lamina propria samples of patients with CD or ulcerative colitis (UC) and healthy individuals (controls). We analyzed the phenotype and functions of the CD4+NKG2D+ T cells and the cytokines they produce in response to NKG2D stimulation. RESULTS: In patients with CD, CD4+ T cells that express NKG2D produced high levels of interleukin (IL)-17 and IL-22 and expressed high levels of CCR6, the IL-23 receptor, CD161, and RORC (a transcription factor that regulates expression of Th17 cytokines). CD4+ T cells that produced IL-17 expressed high levels of NKG2D and CD161. Costimulation of NKG2D and the T-cell receptor (TCR) significantly increased production of IL-17 and tumor necrosis factor α by CD4+ T cells, compared with activation of only the TCR. CD4+NKG2D+ T cells also responded to Th17 polarization. CONCLUSIONS: NKG2D is a functional marker of CD4+ T cells that produce IL-17 in patients with CD, via costimulation of the TCR and NKG2D. Reagents developed to block NKG2D might reduce gastrointestinal inflammation in patients with CD.

Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1.

Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.

Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis.

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.

Rescue of the neuroblastoma mutant of the human nucleoside diphosphate kinase A/nm23-H1 by the natural osmolyte trimethylamine-N-oxide.

The point mutation S120G in human nucleoside diphosphate kinase A, identified in patients with neuroblastoma, causes a protein folding defect. The urea-unfolded protein cannot refold in vitro, and accumulates as a molten globule folding intermediate. We show here that the trimethylamine-N-oxide (TMAO) corrects the folding defect and stimulated subunit association. TMAO also substantially increased the stability to denaturation by urea of both wild-type and S120G mutant. A non-native folding intermediate accumulated in the presence of 4.5-7M urea and of 2M TMAO. It was inactive, monomeric, contained some secondary structure but no tertiary structure and displayed a remarkable stability to denaturation.