Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of na?ve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.     

Genomic,Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1^T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.     

Vaccine-specific local T cell reactivity in immunotherapy-associated vitiligo in melanoma patients

The occurrence of vitiligo in patients with melanoma is especially reported for patients undergoing immunotherapy. While vitiligo in these patients is thought to be related to an immune response directed against melanoma cells, solid evidence is lacking. Here we report local cytotoxic T cell reactivity in three melanoma patients who developed vitiligo, after experimental immunotherapy using dendritic cell vaccinations. Tetramer analysis showed that vaccine-induced T cells recognizing gp100 and tyrosinase are present at the vitiligo lesions. These T cells secrete IFN-γ and IL-2 upon peptide specific stimulation as well as upon recognition of the autologous tumor. We show that functional CD8⁺ T cells specific for melanoma differentiation antigens used in a melanoma immunotherapy trial, do not only invade the tumor, but also the vitiligo lesions. This directly links vitiligo to the immuno-therapeutic intervention and supports the hypothesis that vitiligo is a marker of immunity against melanoma cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reductive dechlorination of beta-hexachlorocyclohexane (beta-HCH) by a Dehalobacter species in coculture with a Sedimentibacter sp

An anaerobic coculture was enriched from a hexachlorocyclohexane (HCH) polluted soil. The coculture reductively dechlorinates the beta-HCH isomer to benzene and chlorobenzene in a ratio of 0.5-2 depending on the amount of beta-HCH degraded. The culture grows with H(2) as electron donor and beta-HCH as electron acceptor, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that the coculture consists of two bacteria that are both related to gram-positive bacteria with a low G + C content of the DNA. One bacterium was identified as a Dehalobacter sp. This bacterium is responsible for the dechlorination. The other bacterium was isolated and characterized as being a Sedimentibacter sp. This strain is not able to dechlorinate beta-HCH. The Dehalobacter sp. requires the presence of Sedimentibacter for growth and dechlorination, but the function of the latter bacterium is not clear. This is the first report on the metabolic dechlorination of beta-HCH by a defined anaerobic bacterial culture.     

NOD2 and toll-like receptors are nonredundant recognition systems of Mycobacterium tuberculosis

Infection with Mycobacterium tuberculosis is one of the leading causes of death worldwide. Recognition of M. tuberculosis by pattern recognition receptors is crucial for activation of both innate and adaptive immune responses. In the present study, we demonstrate that nucleotide-binding oligomerization domain 2 (NOD2) and Toll-like receptors (TLRs) are two nonredundant recognition mechanisms of M. tuberculosis. CHO cell lines transfected with human TLR2 or TLR4 were responsive to M. tuberculosis. TLR2 knock-out mice displayed more than 50% defective cytokine production after stimulation with mycobacteria, whereas TLR4-defective mice also released 30% less cytokines compared to controls. Similarly, HEK293T cells transfected with NOD2 responded to stimulation with M. tuberculosis. The important role of NOD2 for the recognition of M. tuberculosis was demonstrated in mononuclear cells of individuals homozygous for the 3020insC NOD2 mutation, who showed an 80% defective cytokine response after stimulation with M. tuberculosis. Finally, the mycobacterial TLR2 ligand 19-kDa lipoprotein and the NOD2 ligand muramyl dipeptide synergized for the induction of cytokines, and this synergism was lost in cells defective in either TLR2 or NOD2. Together, these results demonstrate that NOD2 and TLR pathways are nonredundant recognition mechanisms of M. tuberculosis that synergize for the induction of proinflammatory cytokines.     

Physiological meaning and potential for application of reductive dechlorination by anaerobic bacteria

Abstract: The physiological meaning of reductive dechlorination reactions catalyzed by anaerobic bacteria can be explained as a co-metabolic activity or as a novel type of respiration. Co-metabolic activities have been found mainly with alkyl halides. They are non-specific reactions catalyzed by various enzyme systems of facultative as well as obligate anaerobic bacteria. In contrast, the reductive dechlorinations involved in metabolic respiration processes are very specific reactions. Only a limited number of alkyl and aryl chlorinated compounds is presently known to function as a terminal electron acceptor in a few, recently isolated bacteria. Metabolic dechlorination rates are in general several orders of magnitude higher than co-metabolic ones. Both reaction types are suitable for the anaerobic treatment of waste streams.     

Enrichment and properties of an anaerobic mixed culture reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-dichlorobenzene.

Hexachlorobenzene (HCB), pentachlorobenzene (QCB), all three isomers of tetrachlorobenzene (TeCB), 1,2,3-trichlorobenzene (1,2,3-TCB), and 1,2,4-TCB were reductively dechlorinated by enrichment cultures in the presence of lactate, glucose, ethanol, or isopropanol as the electron donor. The enrichment cultures originated from percolation columns filled with Rhine River sediment in which dechlorination of TCBs and dichlorobenzenes (DCBs) occurred. A stable consortium obtained by transfer on lactate as the energy and carbon source in the presence of 1,2,3-TCB dechlorinated this isomer stoichiometrically to 1,3-DCB. Dechlorinating activity could only be maintained when an electron donor was added. Lactate, ethanol, and hydrogen appeared to be the best substrates. Optimal temperature and pH for dechlorination were 30 degrees C and 7.2, respectively. The specificity of the enrichment on lactate and 1,2,3-TCB was tested after approximately 60 transfers (after 2.5 years). HCB and QCB were stoichiometrically dechlorinated to 1,3,5-TCB and minor amounts of 1,2,4-TCB. 1,3,5-TCB was the sole product formed from 1,2,3,5-TeCB, while 1,2,3,4-TeCB and 1,2,4,5-TeCB were converted to 1,2,4-TCB. 1,2,4-TCB, 1,3,5-TCB, and the three isomers of DCB were not dechlorinated during 4 weeks of incubation. For further enrichment of the 1,2,3-TCB-dechlorinating bacteria, a two-liquid-phase (hexadecane-water) system was used with hydrogen as the electron donor and 1,2,3-TCB or CO2 as the electron acceptor. Methanogens and acetogens were the major substrate-competing (H2-CO2) microorganisms in the two-liquid-phase system. Inhibition of methanogenesis by 2-bromoethanesulfonic acid did not influence dechlorination, and acetogens which were isolated from the enrichment culture did not have dechlorinating activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment and properties of an anaerobic mixed culture reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-dichlorobenzene.

Hexachlorobenzene (HCB), pentachlorobenzene (QCB), all three isomers of tetrachlorobenzene (TeCB), 1,2,3-trichlorobenzene (1,2,3-TCB), and 1,2,4-TCB were reductively dechlorinated by enrichment cultures in the presence of lactate, glucose, ethanol, or isopropanol as the electron donor. The enrichment cultures originated from percolation columns filled with Rhine River sediment in which dechlorination of TCBs and dichlorobenzenes (DCBs) occurred. A stable consortium obtained by transfer on lactate as the energy and carbon source in the presence of 1,2,3-TCB dechlorinated this isomer stoichiometrically to 1,3-DCB. Dechlorinating activity could only be maintained when an electron donor was added. Lactate, ethanol, and hydrogen appeared to be the best substrates. Optimal temperature and pH for dechlorination were 30 degrees C and 7.2, respectively. The specificity of the enrichment on lactate and 1,2,3-TCB was tested after approximately 60 transfers (after 2.5 years). HCB and QCB were stoichiometrically dechlorinated to 1,3,5-TCB and minor amounts of 1,2,4-TCB. 1,3,5-TCB was the sole product formed from 1,2,3,5-TeCB, while 1,2,3,4-TeCB and 1,2,4,5-TeCB were converted to 1,2,4-TCB. 1,2,4-TCB, 1,3,5-TCB, and the three isomers of DCB were not dechlorinated during 4 weeks of incubation. For further enrichment of the 1,2,3-TCB-dechlorinating bacteria, a two-liquid-phase (hexadecane-water) system was used with hydrogen as the electron donor and 1,2,3-TCB or CO2 as the electron acceptor. Methanogens and acetogens were the major substrate-competing (H2-CO2) microorganisms in the two-liquid-phase system. Inhibition of methanogenesis by 2-bromoethanesulfonic acid did not influence dechlorination, and acetogens which were isolated from the enrichment culture did not have dechlorinating activity.(ABSTRACT TRUNCATED AT 250 WORDS)