Shih-Hsiung Wu

Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA

Indolicidin, a l3-residue antimicrobial peptide-amide, which is unusually rich in tryptophan and proline, is isolated from the cytoplasmic granules of bovine neutrophils. In this study, the structures of indolicidin in 50% D3-trifluoroethanol and in the absence and presence of SDS and D38-dodecylphosphocholine were determined using NMR spectroscopy. Multiple conformations were found and were shown to be due to different combinations of contact between the two WPW motifs. Although indolicidin is bactericidal and able to permeabilize bacterial membranes, it does not lead to cell wall lysis, showing that there is more than one mechanism of antimicrobial action. The structure of indolicidin in aqueous solution was a globular and amphipathic conformation, differing from the wedge shape adopted in lipid micelles, and these two structures were predicted to have different functions. Indolicidin, which is known to inhibit DNA synthesis and induce filamentation of bacteria, was shown to bind DNA in gel retardation and fluorescence quenching experiments. Further investigations using surface plasmon resonance confirmed the DNA-binding ability and showed the sequence preference of indolicidin. Based on our biophysical studies and previous results, we present a diagram illustrating the DNA-binding mechanism of the antimicrobial action of indolicidin and explaining the roles of the peptide when interacting with lipid bilayers at different concentrations.

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD50 increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD50 increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.

The Flexible and Clustered Lysine Residues of Human Ribonuclease 7 Are Critical for Membrane Permeability and Antimicrobial Activity

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 °C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 °C.

Asperjinone, a nor-neolignan, and terrein, a suppressor of ABCG2-expressing breast cancer cells, from thermophilic Aspergillus terreus.

Breast cancer cells express ABCG2 transporters, which mediate multidrug resistance. Discovering a novel compound that can suppress ABCG2 expression and restore drug sensitivity could be the key to improving breast cancer therapeutics. In the current work, one new nor-neolignan, asperjinone (1), as well as 12 other known compounds, was isolated from Aspergillus terreus. The structure of the new isolate was determined by spectroscopic methods. Among these isolates, terrein (2) displayed strong cytotoxicity against breast cancer MCF-7 cells. Treatment with terrein (2) significantly suppressed growth of ABCG2-expressing breast cancer cells. This suppressive effect was achieved by inducing apoptosis via activating the caspase-7 pathway and inhibiting the Akt signaling pathway, which led to a decrease in ABCG2-expressing cells and a reduction in the side-population phenotype.

Isolation and characterization of an active compound from black soybean [Glycine max (L.) Merr.] and its effect on proliferation and differentiation of human leukemic U937 cells.

Black soybean [Glycine max (L.) Merr.] has been used as a health food and herb in China for hundreds of years. In the present study, we purified a unique polysaccharide component from black soybean (PSBS) and found that it indirectly inhibits proliferation and induces differentiation of human leukemic U937 cells via activation of mononuclear cells (MNCs). We prepared conditioned media (MNC-CM) by incubating MNCs from human peripheral blood with or without PSBS (PSBS-MNC-CM and normal MNC-CM, respectively). Treatment of human leukemic U937 cells with PSBS-MNC-CM significantly inhibited proliferation of U937 cells, reducing their growth by 98.5%. Furthermore, PSBS-MNC-CM induced U937 cells to differentiate into mature monocytes/macrophages (83% by morphological examination and 90% by the nitroblue tetrazolium test). Neither PSBS alone nor normal MNC-CM had such effects. The molecular weight of PSBS was about 480 000 by gel filtration. Structural analysis of PSBS revealed that (1,6)-α-D-glucan might be its major active component. Our results suggest that the PSBS may inhibit proliferation and induce differentiation in human leukemic U937 cells by activating the immune response of MNCs.

Lipopolysaccharide O1 Antigen Contributes to the Virulence in Klebsiella pneumoniae Causing Pyogenic Liver Abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganisms ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K1 − O1) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain.

A novel exopolysaccharide from the biofilm of Thermus aquaticus YT-1 induces the immune response through Toll-like receptor 2.

Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2−/− mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.

Efficient and Stereoselective Synthesis of α(2→9) Oligosialic Acids: From Monomers to Dodecamers

most common a(2!8) polysialic acid (1) is found in mammalian tissues and bacteria (Neisseria meningitidis B, Escherichia coli K1, Morexella nonliquefaciens, and Mannheimia haemolytica A2), and the less common a(2!9) polysialic acid (2) and alternating a(2!8)/a(2!9) polysialic acids (3) were discovered to form extracellular capsules of N. meningitidis C and E. coli K92, respectively. Human pathogens encapsulated with polysialic acids cause invasive diseases such as meningitis and urinary tract infections. In pathogenic bacteria, these acidic polysaccharides serve as extracellular shields against the defense systems of their mammalian host. Therefore, polysialic acids are considered good targets for the development of bactericidal agents and antibacterial vaccines. For example, the current vaccines against meningococcal group C diseases are glycoconjugates of isolated a(2!9) polysialic acids and a carrier protein such as diphtheria or tentanus toxoid. However, these kinds of vaccines are often heterogeneous or contaminated with other antigenic components because of the difficulty of purifying polysialic acids from natural sources. An effective method to synthesize pure polysialic acids having a well-defined structure will not only simplify the complexities of vaccines but also provide a better understanding of the structure– activity relationships of polysialic acids in various biological events. Chemical sialylation is complicated as a result of the intrinsic structural features of sialic acid, thus resulting in poor yields or stereoselectivities. Even though notable progress toward the development of sialic acid donors for efficient a sialylation have been reported in the last decade, 12] the synthesis of poly/oligo sialic acid with satisfactory yields and excellent a selectivity is still very challenging. The advancement of donor development led to many approaches for the synthesis of a-specific oligosialic acids, including the synthesis of a(2!9) trisialic acid using C5-azido sialyl phosphite as donor, the synthesis of a(2!9) oligosialic acid using C5-TFA sialyl phosphite as a donor and C5-TFA thiosialoside as an acceptor, and the synthesis of a(2!8) tetrasialoside, a(2!9) trisialoside, and a(2!9) tetrasialoside using 5N,4O-carbonyl-protected thiosialosides. When using 5N,4O-carbonyl-protected thiosialosides as donors, the sequence of assembly starts from the reducing end to the nonreducing end, thus providing an opportunity to stereoselectively elongate the sugar chain one residue at a time. However, this approach has not successfully been used to synthesize an a-specific oligosialic acid polymer that is longer than a tetramer. In principle, convergent block synthesis is an intrinsically better strategy for the preparation of oligomers or polymers and has been applied to the synthesis of some carbohydrate polymers. However, this strategy is hindered by the limited choice of leaving groups to ensure a proper reactivity and Figure 1. Structures of polysialic acids.

Antroquinonol mitigates an accelerated and progressive IgA nephropathy model in mice by activating the Nrf2 pathway and inhibiting T cells and NLRP3 inflammasome

High levels of reactive oxygen species (ROS), systemic T cell activation, and macrophage infiltration in the kidney are implicated in the acceleration and progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis. However, the pathogenic mechanism of IgAN is still little understood, and it remains a challenge to establish a specific therapeutic strategy for this type of glomerular disorder. Recently, we showed that antroquinonol (Antroq), a pure active compound from Antrodia camphorata mycelium, inhibits renal inflammation and reduces oxidative stress in a mouse model of renal fibrosis. But the anti-inflammatory and immune-regulatory effects of Antroq on the acceleration and progression of primary glomerular disorders have not been determined. In this study, we show that Antroq administration substantially impeded the development of severe renal lesions, such as intense glomerular proliferation, crescents, sclerosis, and periglomerular interstitial inflammation, in mice with induced accelerated and progressive IgAN (AcP-IgAN). Further mechanistic analysis in AcP-IgAN mice showed that, early in the developmental stage of the AcP-IgAN model, Antroq promoted the Nrf2 antioxidant pathway and inhibited the activation of T cells and NLRP3 inflammasome. Significantly improved proteinuria/renal function and histopathology in AcP-IgAN mice of an established stage supported potential therapeutic effects of Antroq on the disease. In addition, Antroq was shown to inhibit activation of NLRP3 inflammasome in vitro by an IgA immune complex (IC) partly involving a reduced ROS production in IgA-IC-primed macrophages, and this finding may be helpful in the understanding of the mode of action of Antroq in the treated AcP-IgAN mice.

Structure of human erythrocyte catalase.

Catalase (E.C. 1.11.1.6) was purified from human erythrocytes and crystallized in three different forms: orthorhombic, hexagonal and tetragonal. The structure of the orthorhombic crystal form of human erythrocyte catalase (HEC), with space group P2(1)2(1)2(1) and unit-cell parameters a = 84.9, b = 141.7, c = 232.5 A, was determined and refined with 2.75 A resolution data. Non-crystallographic symmetry restraints were employed and the resulting R value and R(free) were 0.206 and 0.272, respectively. The overall structure and arrangement of HEC molecules in the orthorhombic unit cell were very similar to those of bovine liver catalase (BLC). However, no NADPH was observed in the HEC crystal and a water was bound to the active-site residue His75. Conserved lattice interactions suggested a common growth mechanism for the orthorhombic crystals of HEC and BLC.