The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB 2 A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane ␣-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes.Multiple drug resistant bacteria poses a threat to man's fight against infectious diseases. Some multiple drug resistance systems may detoxify their substrates by transport across the complete cell wall of Gram-negative bacteria, across cytoplasmic membrane, periplasm, and outer membrane. These assumed transenvelope transporters are composed of a pump protein that energizes the transport, in addition to a membrane fusion and an outer membrane-associated protein (1, 2). The pump protein may be an ATP-binding cassette transporter (3, 4), a transporter of the major facilitator superfamily (5), or a resistance-nodulation-cell division (RND) 1 protein (4, 6, 7). The archetype of the RND permease superfamily family is CzcA from the Gram-negative bacterium Ralstonia sp. CH34 (formerly Alcaligenes eutrophus strain CH34) (8 -12).This bacterium contains at least seven heavy metal resistance determinants, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (163 kilobase pairs) and pMOL30 (238 kb) (8, 13-16). One of them, the czc-determinant of plasmid pMOL30, mediates inducible resistance to millimolar concentrations of Co 2ϩ , Zn 2ϩ , and Cd 2ϩ in strain CH34 (8, 17). The products of the genes czcA, czcB, and czcC form a membrane-bound protein complex catalyzing an energy-dependent efflux of these three metal cations (9, 11), probably across the complete envelope. The mechanism of action of CzcCB 2 A is that of a proton/cation antiporter, and the K m values of the efflux system for the substrate heavy metal cations are also in the millimolar range (10). Although indirect evidence led to the assumption that CzcA is the central cation/proton antiporter of the CzcCB 2 A complex (10), this has not been shown directly. This paper demonstrates that CzcA is a cation/proton antiporter, and develops the model of CzcA as a two-channel pump based on topology studies and the function of CzcA mutant proteins. This model sheds some light on other RND proteins involved in multiple drug resistance of bacteria or with previously unknown functions in mammals. EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Growt...

Background:The current structural model of Streptococcus pneumoniae lipoteichoic acid reveals inconsistencies. Results: High resolution NMR and MS analysis of O-deacylated pnLTA allowed a precise revision of its structure. Conclusion:The novel structure presented here is in complete agreement with known structural, biosynthetic, and immunological data. Significance: This study will aid in further understanding the biosynthesis and inflammatory potency of pneumococcal (lipo)teichoic acids.

The respiratory pathogen Streptococcus pneumoniae has evolved efficient mechanisms to resist oxidative stress conditions and to displace other bacteria in the nasopharynx. Here we characterize at physiological, functional and structural levels two novel surface-exposed thioredoxin-family lipoproteins, Etrx1 and Etrx2. The impact of both Etrx proteins and their redox partner methionine sulfoxide reductase SpMsrAB2 on pneumococcal pathogenesis was assessed in mouse virulence studies and phagocytosis assays. The results demonstrate that loss of function of either both Etrx proteins or SpMsrAB2 dramatically attenuated pneumococcal virulence in the acute mouse pneumonia model and that Etrx proteins compensate each other. The deficiency of Etrx proteins or SpMsrAB2 further enhanced bacterial uptake by macrophages, and accelerated pneumococcal killing by H2O2 or free methionine sulfoxides (MetSO). Moreover, the absence of both Etrx redox pathways provokes an accumulation of oxidized SpMsrAB2 in vivo. Taken together our results reveal insights into the role of two extracellular electron pathways required for reduction of SpMsrAB2 and surface-exposed MetSO. Identification of this system and its target proteins paves the way for the design of novel antimicrobials.

Two human Golli (for gene expressed in the oligodendrocyte lineage)-MBP (for myelin basic protein) cDNAs have been isolated from a human oligodendroglioma cell line. Analysis of these cDNAs has enabled us to determine the entire structure of the human Golli-MBP gene. The Gofli-MBP gene, which encompasses the MBP transcription unit, is =179 kb in length and consists of 10 exons, seven of which constitute the MBP gene. The human Goli-MBP gene contains two transcription start sites, each of which gives rise to a family of alternatively spliced transcripts. At least two Golli-MBP transcripts, containing the first three exons ofthe gene and one or more MBP exons, are produced from the first transcription start site. The second family of transcripts contains only MBP exons and produces the well-known MBPs. In humans, RNA blot analysis revealed that Golli-MBP transcripts were expressed in fetal thymus, spleen, and human B-cell and macrophage cell lines, as well as in fetal spinal cord. These rmdings clearly link the expression of exons encoding the autoimmunogen/encephalitogen MBP in the central nervous system to cells and tissues of the immune system through normal expression of the Golli-MBP gene. They also establish that this genetic locus, which includes the MBP gene, is conserved among species, providing further evidence that the MBP transcription unit is an integral part of the Golli transcription unit and suggest that this structural arrangement is important for the genetic function and/or regulation of these genes.

The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport. The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit. The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation. Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter. Fusions of CzcC and CzcB with alkaline phosphatase and ␤-galactosidase are in agreement with a periplasmic location of most parts of both proteins. Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein. Based on these data, a new working model for the function of the Czc system is discussed.Alcaligenes eutrophus CH34 contains at least seven heavy metal resistance determinants, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (163 kb) and pMOL30 (238 kb) (9,15,17,19,20). One of them, the czc determinant of plasmid pMOL30, mediates inducible resistance to Co 2ϩ , Zn 2ϩ , and Cd 2ϩ in A. eutrophus CH34 (21). The products of the genes czcA, czcB, and czcC form a membrane-bound protein complex catalyzing an energy-dependent efflux of these three metal cations (25,26). The mechanism of action of CzcCBA is that of a cation-proton antiporter (24).Besides the CzcR and CzcS regulatory proteins (37), the membrane-bound CzcD, possibly a sensor protein, is essential for induction of czc (23). CzcD belongs to the newly coined CDF (cation diffusion facilitator) family (27). The function of the products of additional czc genes located upstream of the czcCBA structural gene region remains unclear (37).In an old working model (22,27), CzcA may function as a cation-proton antiporter and CzcB may function as a cationbinding subunit; both subunits together form the Zn 2ϩ efflux system. The CzcC protein acts as a modifier extending the substrate specificity to Co 2ϩ and Cd 2ϩ . This model is based on computer-assisted predictions of the secondary structure and membrane orientation of the three proteins CzcA, CzcB, and CzcC and on the analysis of deletion mutants (25). In this study, we reinvestigated the function and localization of the three subunits by using more sophisticated approaches, and here we propose a new working model. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. A. eutrophus AE104 (17) is a metal-sensitive, plasmid-free derivative of strain CH34. Escherichia coli K38(pGP1-2) (36) was used for expression of czcCBAD derivatives under control of the phage T7 promoter as described previously (19). The other bacteria...

The myelin basic protein (MBP) gene is part of the golli-mbp gene complex. In mouse, the golli-mbp gene produces two families of mRNAs from different transcription start sites that generate either MBPs or golli proteins (which contain MBP sequences in addition to unique peptide sequences). In situ hybridization and immunocytochemical analyses indicate that golli products are expressed in selected neuronal populations in postnatal mouse brain, in addition to oligodendrocytes, as shown earlier. The principal subcellular location of golli proteins in neurons was in axonal and dendritic processes. In a small subset of neurons, golli proteins were located in nuclei. With development and neuronal maturation, golli-mbp expression decreased and/or there was a striking shift in subcellular localization from nuclei and cell soma to the cell processes in specific neuronal populations. Golli protein was localize in neurites of migrating cerebellar granule cells, but it shifted to a nuclear localization when the cells took up residence in the internal granule cell layer. In some regions, (e.g., olfactory bulb and cerebellum) golli proteins were expressed over the entire postnatal period examined (birth to 75 d). The unique patterns of developmental expression within individual populations of neurons, and the unusual shift in subcellular localization of golli proteins with neuronal migration and maturation, suggest a complex regulation of this gene at both the transcriptional and posttranslational levels. The data also suggest that the cellular function(s) of the golli proteins is very different from the structurally related MBPs.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. In this study, we examine an innate immune recognition pathway that senses pneumococcal infection, triggers type I IFN production, and regulates RANTES production. We found that human and murine alveolar macrophages as well as murine bone marrow macrophages, but not alveolar epithelial cells, produced type I IFNs upon infection with S. pneumoniae. This response was dependent on the pore-forming toxin pneumolysin and appeared to be mediated by a cytosolic DNA-sensing pathway involving the adapter molecule STING and the transcription factor IFN regulatory factor 3. Indeed, DNA was present in the cytosol during pneumococcal infection as indicated by the activation of the AIM2 inflammasome, which is known to sense microbial DNA. Type I IFNs produced by S. pneumoniae-infected macrophages positively regulated gene expression and RANTES production in macrophages and cocultured alveolar epithelial cells in vitro. Moreover, type I IFNs controlled RANTES production during pneumococcal pneumonia in vivo. In conclusion, we identified an immune sensing pathway detecting S. pneumoniae that triggers a type I IFN response and positively regulates RANTES production.

The Czc system of Ralstonia sp. strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. The CzcD protein is involved in the regulation of the Czc system. It is a membrane-bound protein with at least four transmembrane α-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life. The deletion ofczcD in a Ralstonia sp. led to partially constitutive expression of the Czc system due to an increased transcription of the structural czcCBA genes, both in the absence and presence of inducers. The czcD deletion could be fully complemented in trans by CzcD and two other CDF proteins from Saccharomyces cerevisiae, ZRC1p and COT1p. All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in Ralstonia, and this resistance was based on a reduced accumulation of the cations. Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.