Characterization of the Structure and Biological Functions of a Capsular Polysaccharide Produced by Staphylococcus saprophyticus

Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, SCC_15305RM and SCC_15305cap. The SCC_15305cap element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK (capD_Ssp, capL_Ssp, and capK_Ssp) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and capI_Ssp show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists.Approximately 13 million women develop urinary tract infections (UTIs) annually in the United States, with a recurrence rate between 25% and 44% (45). Staphylococcus saprophyticus is second only to Escherichia coli as a cause of uncomplicated UTI in young women (45, 46). A novobiocin-resistant member of the coagulase-negative staphylococci (60), S. saprophyticus has rarely exhibited resistance to other antibiotics (25). However, a recent report (19) indicated that methicillin-resistant S. saprophyticus isolates have emerged in Japan. The gastrointestinal tract and the vagina are the major reservoirs of S. saprophyticus (18, 30) and the likely sources of recurrent infection (20, 37, 49). Approximately 40% of patients with S. saprophyticus UTI present with acute pyelonephritis (22, 30). These patients experience symptoms more severe than those of patients infected by E. coli (24), and they are more likely to develop recurrent infections (21).A number of potential virulence factors have been identified in S. saprophyticus. Gatermann et al. showed that in a rodent model of ascending UTI, the production of urease contributes to S. saprophyticus growth and pathogenicity in the bladder (10, 12). Other putative virulence factors of S. saprophyticus include a surface-associated lipase (11, 51, 53), the collagen binding protein SdrI (52), and a cell wall-anchored hemagglutinin protein that mediates the binding of S. saprophyticus to sheep erythrocytes, fibronectin, and human uroepithelial cells (14, 29, 34, 35). The hemagglutinin was dubbed UafA in the sequenced ATCC 15305 strain, and deletion of the uafA gene resulted in reduced S. saprophyticus hemagglutination (HA) and adherence to human bladder carcinoma cells (29). Kuroda et al. noted that UafA-mediated adherence of S. saprophyticus to the T24 cell line was inhibited by the presence of the ATCC 15305 polysaccharide capsule (29).Staphylococcal species produce a variety of extracellular glycopolymers that contribute to the surface properties and virulence of the bacterium, such as capsular polysaccharides (CP), teichoic acids, and poly-N-acetylglucosamine (PNAG). CP production renders Staphylococcus aureus resistant to opsonophagocytic killing; alanine modifications of teichoic acids promote bacterial resistance to antimicrobial peptides (40); and PNAG is involved in biofilm formation (4). Recently, the secretion of another anionic polymer (poly-γ-dl-glutamic acid) by certain other coagulase-negative staphylococci was reported (28). Polyglutamic acid production is enhanced under high-salt conditions and may contribute to the survival of Staphylococcus epidermidis on human skin.S. saprophyticus strain 15305 does not produce PNAG or polyglutamic acid (28, 29), but this uropathogenic species is encapsulated. CP are lacking in isolates of S. epidermidis, the most common of the coagulase-negative species, but genomic evidence indicates that Staphylococcus haemolyticus (7, 57), S. saprophyticus (29), and Staphylococcus carnosus (47) carry capsule loci with genetic similarity to the Staphylococcus aureus cap5 (cap8) gene locus. In this study, we purified and characterized the CP produced by S. saprophyticus ATCC 15305 and investigated the CP phenotype of S. saprophyticus clinical isolates.     

Pulmonary Collectins Protect Macrophages against Pore-forming Activity of Legionella pneumophila and Suppress Its Intracellular Growth

Pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate immunity of the lung. Legionella pneumophila is a bacterial respiratory pathogen that can replicate within macrophages and causes opportunistic infections. L. pneumophila possesses cytolytic activity, resulting from insertion of pores in the macrophage membrane upon contact. We examined whether pulmonary collectins play protective roles against L. pneumophila infection. SP-A and SP-D bound to L. pneumophila and its lipopolysaccharide (LPS) and inhibited the bacterial growth in a Ca²⁺-dependent manner. The addition of LPS in the culture blocked the inhibitory effects on L. pneumophila growth by the collectins, indicating the importance of LPS-collectin interaction. When differentiated THP-1 cells were infected with L. pneumophila in the presence of SP-A and SP-D, the number of permeable cells was significantly decreased, indicating that pulmonary collectins inhibit pore-forming activity of L. pneumophila. The number of live bacteria within the macrophages on days 1–4 after infection was significantly decreased when infection was performed in the presence of pulmonary collectins. The phagocytosis experiments with the pH-sensitive dye-labeled bacteria revealed that pulmonary collectins promoted bacterial localization to an acidic compartment. In addition, SP-A and SP-D significantly increased the number of L. pneumophila co-localized with LAMP-1. These results indicate that pulmonary collectins protect macrophages against contact-dependent cytolytic activity of L. pneumophila and suppress intracellular growth of the phagocytosed bacteria. The promotion of lysosomal fusion with Legionella-containing phagosomes constitutes a likely mechanism of L. pneumophila growth suppression by the collectins.     

Chirality of camphor derivatives by density functional theory

Infrared (IR) and vibrational circular dichroism (VCD) spectra of chiral camphor, camphorquinone and camphor-10-sulfonic acid (CSA), known as standard compounds for electronic circular dichroism (ECD) spectroscopy, are measured and their vibrational frequencies, infrared intensities, and rotational strengths are calculated using density functional theory (DFT). The observed IR and VCD spectra of chiral camphor and camphorquinone in carbon tetrachloride solution are reproduced by the DFT calculations, but those of CSA are not. DFT calculations of hydration models, where an anionic CSA specifically binds a few water molecules, are carried out. The average of the simulated VCD spectra in the hydration models is more consistent with the observed spectra. In addition, the wavelengths and dipole and rotational strengths for chiral camphor, camphorquinone, anionic CSA, and the hydration models were calculated by time-dependent DFT. In the region of 280-300 nm, the calculated wavelengths of the ECD bands for chiral camphor and camphorquinone coincide with the observed wavelengths that have been reported, and the calculated wavelengths for the hydration models are closer to the observed wavelengths reported than are those calculated for chiral anionic CSA. Consequently, the analysis combined with VCD and ECD spectroscopy using DFT calculations can elucidate the chirality of optically active molecules, even in an aqueous solution.     

Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.     

Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.     

Far-Field Focusing of Spiral Plasmonic Lens

In this paper, we study the nanoscale-focusing effect in the far field for a spiral plasmonic lens with a concentric annular groove by using finite-difference time domain simulation. The simulation result demonstrates that a left-hand spiral plasmonic lens can concentrate an incident right-hand circular polarization light into a focal spot at the exit surface. And this spot can be focused into far field due to constructive interference of the scattered light by the annular groove. The focal length and the focal depth can be adjusted by changing the groove radius and number of grooves within a certain range. These properties make it possible to probe the signal of spiral plasmonic lens in far field by using conventional optical devices.     

Proteasome-dependent degradation of alpha-catenin is regulated by interaction with ARMc8alpha

ARMc8 (armadillo-repeat-containing protein 8) is a key component of the CTLH (C-terminal to lissencephaly type-1-like homology motif) complex in mammalian cells. This complex is well conserved in Saccharomyces cerevisiae and has been characterized as a FBPase (fructose-1, 6-bisphosphatase)-degrading complex. The yeast homologue of ARMc8, Gid (glucose-induced degradation) 5p, plays an essential role in the ubiquitin- and proteasome-dependent degradation of FBPase. To elucidate the function of ARMc8, we used a yeast two-hybrid system to screen a human skeletal muscle cDNA library. alpha-Catenin was isolated as a binding protein of ARMc8alpha. This association was confirmed by co-immunoprecipitation assay using MDCK (Madin-Darby canine kidney) cells in which exogenous alpha-catenin and ARMc8alpha were overexpressed. The association was also confirmed by co-immunoprecipitation assay using endogenous proteins in untransfected MDCK cells. We then used immunofluorescence microscopy of MDCK cells and C2C12 cells to investigate the intracellular distribution of ARMc8. Exogenously expressed ARMc8 was co-localized with alpha-catenin and beta-catenin along the cell membrane, suggesting an association between alpha-catenin and ARMc8 in the cells. To compare the binding domain of alpha-catenin with ARMc8alpha with that of beta-catenin, we performed a co-immunoprecipitation assay, again using 5'- and 3'-deletion constructs of alpha-catenin. The N-terminal sequence (amino acids 82-148) of alpha-catenin was sufficient to bind to both ARMc8alpha and beta-catenin. Next, we investigated the proteasome-dependent degradation of alpha-catenin by immunoblotting using proteasome inhibitors. Co-expression of ARMc8alpha with alpha-catenin resulted in rapid degradation of the exogenous alpha-catenin. Furthermore, ARMc8 knockdown inhibited alpha-catenin degradation and prolonged the half-life of alpha-catenin. We conclude that ARMc8alpha associates with alpha-catenin and up-regulates its degradation.     

Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain

Pulmonary surfactant protein D (SP-D) is a member of the collectin family and plays crucial roles in the innate immunity of the lung. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with MD-2 and alters lipopolysaccharide signaling. In this study, we examined and characterized the binding of SP-D to MD-2 using a soluble form of recombinant MD-2 (sMD-2). SP-D bound in a concentration- and Ca(2+)-dependent manner to sMD-2 coated onto microtiter wells. Excess mannose abolished the binding of SP-D to sMD-2. In solution, SP-D cosedimented with sMD-2 in the presence of Ca(2+). The direct binding of SP-D to sMD-2 was confirmed by BIAcore analysis. Anti-SP-D monoclonal antibody that recognizes the carbohydrate recognition domain (CRD) of SP-D significantly inhibited the binding of SP-D to sMD-2, indicating the involvement of the CRD for the binding to sMD-2. Ligand blot analysis revealed that SP-D bound to N-glycopeptidase F-treated sMD-2. In addition, the biotinylated SP-D pulled down the mutant sMD-2 with Asn(26) --> Ala and Asn(114) --> Ala substitutions, which lacks the consensus for N-glycosylation. Furthermore, the sMD-2 mutant cosedimented SP-D. These results demonstrate that SP-D directly interacts with MD-2 through the CRD.     

Existence of beta-methylnorleucine in recombinant hirudin produced by Escherichia coli.

A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli, which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l(-1)) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as beta-methylnorleucine (beta MeNle; (2S,3S)-2-amino-3-methylhexanoic acid). The modification pathway of beta MeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, beta MeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of beta MeNle in E. coli, which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.