Molecular characterization of erm(B)- and mef(E)-mediated erythromycin-resistant Streptococcus pneumoniae in China and complete DNA sequence of Tn2010

Aims: To characterize the erm(B)‐ and mef(E)‐mediated erythromycin‐resistant Streptococcus pneumoniae clinical isolates obtained from ten hospitals located different cities in China. Methods and Results: Totally 83 S. pneumoniae were collected, and eighteen representative strains of 66 strains that exhibited erythromycin resistance were used for further characterization by antibiograms, serotyping, PFGE, MLST, DNA sequencing of the macrolide‐resistance elements and mapping of the elements on the chromosome. Twelve isolates showed a high‐level resistance to erythromycin, and six other isolates showed a low‐level resistance to erythromycin. Thirteen isolates harboured a Tn2010 transposon (26·4 kbp) encoding the erm(B), tet(M) and mef(E) genes and were classified into three types by Tn2010 structures. The remaining five isolates harboured a Tn6002 transposon (20·9 kbp) encoding the erm(B) and tet(M) genes and were classified into three types by Tn6002 locations on the chromosome. Three of the Tn6002 elements were located within the Tn5252‐like element, implying that these composed a large mobile element. The MLST analyses showed that several clones had been disseminated and that the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Four new MLST strains, which were designated as ST3262, ST3263, ST3397 and ST3398 were also identified. Conclusions: The erythromycin resistance determinant of S. pneumoniae that had been isolated in China was located in Tn2010 or the Tn6002 element and several clones had been disseminated, and the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Significance and Impact of the Study: This is the first molecular analysis of erythromycin‐resistant Streptococcus pneumoniae clinical isolates in China, and the first report of the complete nucleotide sequence of Tn2010 (26 390 bp).     

黏蛋白型O-聚糖：结构、功能及与肿瘤的相关性

黏蛋白是细胞表面的或分泌的、具有高度O-糖基化修饰的糖蛋白。黏蛋白型O-聚糖是由多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T）家族催化起始合成的,在肿瘤中常常伴随着黏蛋白型O-聚糖结构和数量上的改变,形成肿瘤特异聚糖结构（cancer-associated glycans）,如肿瘤Tn和T抗原等。肿瘤特异聚糖使肿瘤细胞的抗原性和黏附能力发生改变,促进肿瘤细胞的恶性增生与转移。而这些肿瘤特异聚糖结构,也为肿瘤的诊断与抗肿瘤药物或疫苗开发提供了理论基础。     

Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.     

Evidence for the location of the allosteric activation switch in the multisubunit phosphorylase kinase complex from mass spectrometric identification of chemically crosslinked peptides

Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.     

Sweet escape: Sialic acids in tumor immune evasion

Sialic acids represent a family of sugar molecules derived from neuraminic acid that frequently terminate glycan chains and contribute to many biological processes. Already five decades ago, aberrantly high expression of sialic acids has been proposed to protect cancer cells from recognition and eradication by the immune system. Today, increased understanding at the molecular level demonstrates the broad immunomodulatory capacity of tumor-derived sialic acids that is, at least in part, mediated through interactions with immunoinhibitory Siglec receptors. Here we will review current studies from a sialic acid sugar perspective showing that tumor-derived sialic acids disable major killing mechanisms of effector immune cells, trigger production of immune suppressive cytokines and dampen activation of antigen-presenting cells and subsequent induction of anti-tumor immune responses. Furthermore, strategies to modulate sialic acid expression in cancer cells to improve cancer immunotherapy will be discussed.     

Familial hypertrophic cardiomyopathy related E180G mutation increases flexibility of human cardiac α-tropomyosin

α-Tropomyosin (αTm) is central to Ca²⁺-regulation of cardiac muscle contraction. The familial hypertrophic cardiomyopathy mutation αTm E180G enhances Ca²⁺-sensitivity in functional assays. To investigate the molecular basis, we imaged single molecules of human cardiac αTm E180G by direct probe atomic force microscopy. Analyses of tangent angles along molecular contours yielded persistence length corresponding to ∼35% increase in flexibility compared to wild-type. Increased flexibility of the mutant was confirmed by fitting end-to-end length distributions to the worm-like chain model. This marked increase in flexibility can significantly impact systolic and possibly diastolic phases of cardiac contraction, ultimately leading to hypertrophy.     

Ca2+ -induced tropomyosin movement in scallop striated muscle thin filaments

Striated muscle contraction in most animals is regulated at least in part by the troponin-tropomyosin (Tn-Tm) switch on the thin (actin-containing) filaments. The only group that has been suggested to lack actin-linked regulation is the mollusks, where contraction is regulated through the myosin heads on the thick filaments. However, molluscan gene sequence data suggest the presence of troponin (Tn) components, consistent with actin-linked regulation, and some biochemical and immunological data also support this idea. The presence of actin-linked (in addition to myosin-linked) regulation in mollusks would simplify our general picture of muscle regulation by extending actin-linked regulation to this phylum as well. We have investigated this question structurally by determining the effect of Ca²⁺ on the position of Tm in native thin filaments from scallop striated adductor muscle. Three-dimensional reconstructions of negatively stained filaments were determined by electron microscopy and single-particle image analysis. At low Ca²⁺, Tm appeared to occupy the “blocking” position, on the outer domain of actin, identified in earlier studies of regulated thin filaments in the low-Ca²⁺ state. In this position, Tm would sterically block myosin binding, switching off filament activity. At high Ca²⁺, Tm appeared to move toward a position on the inner domain, similar to that induced by Ca²⁺ in regulated thin filaments. This Ca²⁺-induced movement of Tm is consistent with the hypothesis that scallop thin filaments are Ca²⁺ regulated.     

途径工程及Tn5转座子介导突变提高大肠杆菌丙酮酸生产

为了开发丙酮酸高产菌株,以大肠杆菌MG1655为出发菌株,通过基因敲除阻断副产物途径构建了产丙酮酸大肠杆菌工程菌KLPP。进一步利用p UT Mini-Tn5载体进行转座子随机突变,构建了含有7 197个单克隆的突变体文库。使用基于丙酮酸的二硝基苯肼显色法,建立了96孔板-酶标仪快速筛选方法,经过两轮的筛选,成功筛选到了6个突变体菌株,比KLPP丙酮酸产量提高了38%、31%、19%、28%、44%和14%。利用全基因组重测序确定了其转座子插入的位置,进而确定了可能影响丙酮酸产量的基因位点,为后续菌株改造工作奠定了基础。     

Characterization of the molybdate transport system ModABC of Staphylococcus carnosus

Transposon mutagenesis of Staphylococcus carnosus led to the identification of three genes, modABC, which encode an ABC transporter that is involved in molybdate transport. It was shown by [¹⁴C]palmitate labeling that ModA represents a lipoprotein that in gram-positive bacteria is the counterpart of the periplasmic binding proteins of gram-negative organisms. The sequence characteristics identify ModB as the integral-membrane, channel-forming protein and ModC as the ATP-binding energizer for the transport system. Mutants defective in modABC had only 0.4% of the wild-type nitrate reductase activity. Molybdate at a non-physiologically high concentration (100 μM) fully restored nitrate reductase activity, suggesting that at least one other system is able to transport molybdate, but with lower affinity. The expression of modA (and most likely of modBC) was independent of oxygen and nitrate. To date, there are no indications for molybdate-specific regulation of modABC expression since in a modB mutant, modA expression was unchanged in comparison to the wild-type. Received: 5 February 1999 / Accepted: 31 May 1999