Biochemical characterization of the surface-associated lipase of Staphylococcus saprophyticus

Staphylococcus saprophyticus, an important cause of urinary tract infections, produces a surface-associated lipase, Ssp. In contrast to other lipases, Ssp is a protein that is present in high amounts on the surface of the bacteria and it was shown that it is a true lipase. Characterization of S. saprophyticus lipase (Ssp) showed that it is more similar to Staphylococcus aureus lipase and Staphylococcus epidermidis lipase than to Staphylococcus hyicus lipase and Staphylococcus simulans lipase. Ssp showed an optimum of lipolytic activity at pH 6 and lost its activity at pH>8 or pH<5. The present results show that Ssp activity is dependent on Ca(2+). Consequently, activity increased c. 10-fold in the presence of 2 mM Ca(2+). Optimal activity was reached at 30 degrees C. It was also observed that the enzymatic activity of Ssp depends strongly on the acyl chain length of the substrate molecule.     

The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of d-serine

Human urine contains a relatively high concentration of d -serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d -serine deaminase gene ( dsd A) of UPEC. We found the gene in several clinical isolates of S. saprophyticus ; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii , phylogenetically close relatives of S. saprophyticus , and could also not be detected in isolates of Staphylococcus aureus , Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d -serine, whereas S. aureus , S. epidermidis and other staphylococcal species could not. The association of the dsd A gene with growth in media including d -serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d -serine utilization and detoxification may be a general property of uropathogenic bacteria.     

Cloning of an agr homologue of Staphylococcus saprophyticus

An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.     

Time-based distribution of Staphylococcus saprophyticus pulsed field gel-electrophoresis clusters in community-acquired urinary tract infections

The epidemiology of urinary tract infections (UTI) by Staphylococcus saprophyticus has not been fully characterised and strain typing methods have not been validated for this agent. To evaluate whether epidemiological relationships exist between clusters of pulsed field gel-electrophoresis (PFGE) genotypes of S. saprophyticus from community-acquired UTI, a cross-sectional surveillance study was conducted in the city of Rio de Janeiro, Brazil. In total, 32 (16%) female patients attending two walk-in clinics were culture-positive for S. saprophyticus. Five PFGE clusters were defined and evaluated against epidemiological data. The PFGE clusters were grouped in time, suggesting the existence of community point sources of S. saprophyticus. From these point sources, S. saprophyticus strains may spread among individuals.     

Hemagglutinating and adherence properties of Staphylococcus saprophyticus: epidemiology and virulence in experimental urinary tract infection of rats

Abstract We studied hemagglutinating and adherence properties in Staphylococcus saprophyticus isolates originating from symptomatic urinary tract infections. 12/13 (92%) of strains adhered to Hep cells and 11/13 (85%) were able to agglutinate sheep erythrocytes. Adherence properties differed markedly between strains ( P < 0.0001. Two strains, one hemagglutinating and adherent and one negative for both properties were chosen for experimental urinary tract infections. Results indicate that presence of the hemagglutinin favours colonization of kidney tissue.     

Staphylococcus saprophyticus ATCC 15305 is internalized into human urinary bladder carcinoma cell line 5637

Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.     

Agglutination of Bordetella species by lectins

Phase I cells of Bordetella pertussis but not those of B. parapertussis, B. bronchiseptica or B. avium were agglutinated by Limulus polyphemus lectin. Most strains of B. pertussis but not those of the other species were also agglutinated by Helix pomatia lectin. In precipitation reactions between lectins and purified Bordetella lipopolysaccharide (LPS) preparations a similar pattern occurred. Lectin agglutination provides a rapid presumptive method for the differentiation of B. pertussis from B. parapertussis and other Bordetella species.     

Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding

Staphylococci use cell wall‐anchored proteins as adhesins to attach to host tissues. Staphylococcus saprophyticus, a uropathogenic species, has a unique cell wall‐anchored protein, uro‐adherence factor A (UafA), which shows erythrocyte binding activity. To investigate the mechanism of adhesion by UafA, we determined the crystal structure of the functional region of UafA at 1.5 Å resolution. The structure was composed of three domains, designated as the N2, N3, and B domains, arranged in a triangular relative configuration. Hemagglutination inhibition assay with domain‐truncated mutants indicated that both N and B domains were necessary for erythrocyte binding. Based on these results, a novel manner of ligand binding in which the B domain acts as a functional domain was proposed as the adhesion mechanism of S. saprophyticus.     

腐生葡萄球菌M36耐有机溶剂脂肪酶基因的克隆与原核表达

脂肪酶是重要的工业用酶,在食品加工、生物柴油的合成等领域具有广泛的应用。但是在应用中有机溶剂对脂肪酶具有一定的毒性,因此获得耐有机溶剂的脂肪酶基因并实现高效表达是脂肪酶规模化应用的前提。本研究应用PCR技术首次从耐有机溶剂脂肪酶产生菌腐生葡萄球菌M36基因组DNA中扩增得到脂肪酶Ⅲ基因lip3(GenBank AccessionNo.FJ979867),其编码区长度为741bp,编码247个氨基酸,推测蛋白分子量大小为31.6kD。它与腐生葡萄球菌lip3推测的基因(GenBank AccessionNo.AP008934)只有83%的同源性。将该基因与大肠杆菌表达载体pET-DsbA连接,转化大肠杆菌EscherichiacoliBL21(DE3)获得重组菌株BL21(DE3)/pET-DsbA-lip3,在pH8、25oC条件下,OD600为1.0时用0.4mmol/LIPTG诱导12h酶活达到25.8U/mL。重组酶在甲醇、正己烷、异辛烷、正庚烷等有机溶剂中具有较好的耐性。lip3基因的克隆及在大肠杆菌中有效表达的研究为进一步进行基因工程改造和脂肪酶应用奠定了基础。     

Divalent cation enhancement of the agglutinability by soyabean lectin of liposomes prepared from total lipid of erythrocytes and of erythrocyte membranes

CaCl₂ or MgCl₂ but not NaCl enhances the soyabean lectin-induced agglutination of liposomes prepared from total lipids of erythrocyte membranes. The addition of purified phosphatidylserine to the total lipids of erythrocyte membranes before the formation of liposomes inhibits lectin-induced agglutinability of the preparation in the absence of CaCl₂, but not in its presence. When preformed phosphatidylserine liposomes are added to liposomes of total lipids of erythrocyte ghosts, they do not inhibit agglutination, indicating that phosphatidylserine does not inhibit the lectin directly. CaCl₂ or MgCl₂ but not NaCl also stimulates the soyabean lectin-induced agglutination of human erythrocyte membranes.Electron micrographs indicate that the liposome preparations are multilamellar and separate even in the presence of CaCl₂. When such liposomes are treated with lectin with or without CaCl₂, the electron micrographs show significant agglutination without apparent fusion. The reversal of the agglutination of liposomes by specific sugars followed by turbidimetric and electron microscopic techniques supports the conclusion that CaCl₂ stimulated lectin-induced agglutination is unaccompanied by fusion.The stimulation by divalent cations of lectin-induced agglutination of erythrocyte ghosts or of our liposomes may be due to a decrease in apparent surface charge of these membrane systems.     

Urease from Staphylococcus saprophyticus: purification,characterization and comparison to Staphylococcus xylosus urease

Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420000; it consisted of subunits with molecular weights of 72400 (), 20400 (), and 13900 () in an estimated ()₄ stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420000 to 100000. In the native enzyme, 4.09 (±0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.Abbreviation PAGE polyacrylamide gel electrophoresis     

Adhesion of Staphylococcus epidermidis and Staphylococcus saprophyticus to a hydrophobic biomaterial

The relative surface charge and hydrophobicity of 16 strains of Staphylococcus epidermidis showed large variations. For this species no relationship between the two surface parameters was found. A highly negative surface charge was observed in all seven encapsulated strains (one S. epidermidis and six Staphylococcus saprophyticus strains). The adhesion of the staphylococci to fluorinated polyethylene-propylene films was not related to the relative surface charge and the hydrophobicity of the bacteria. On films pre-exposed to human plasma, the bacterial adhesion was substantially reduced. Mechanisms involved in the adhesion of coagulase-negative staphylococci to this biomaterial are discussed.     

A lectin cytochemical study of glycoconjugates in the human retina

Summary The binding to morphologically normal human retina of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities was studied. Eight formalin-fixed and paraffin-embedded eyes were examined. Photoreceptor cells bound Lens culinaris (LCA), wheat germ (WGA), peanut (PNA) and Ricinus communis (RCAI) agglutinins, and concanavalin A (ConA). The outer segment region was labeled more strongly than the inner segment region, and PNA labeled only cones. All these lectins except PNA bound to both plexiform layers, and all but PNA and RCAI to the nuclear layers. Pretreatment with neuraminidase to remove sialic acid resulted in increased binding of RCAI and PNA, which now labeled both rods and cones, and in decreased binding of WGA. Bandeiraea simplicifolia (BSAI), Dolichos biflorus (DBA), soybean (SBA), Ulex europaeus (UEAI), and Lotus tetragonolobus (LTA) agglutinins, as well as pokeweed mitogen (PWM) reacted only with retinal vascular endothelial cells, which were also labeled with the other lectins. The results indicate that -mannose, -glucose, -galactose, N-acetyl-d-glucosamine and N-acetylneuraminic acid are present in glycoconjugates of human neuroretina.     

Estimation of Microorganism Counts in Agglutination and Enzyme Linked Immuno‐Sorbent Assays: Consequences of Ignoring Censored Data

The consequences of ignoring censored data on the estimation of mean microorganism counts using agglutination assays or certain classes of enzyme linked immuno‐sorbent assays are examined. It is shown that both overestimation and underestimation of the maximum likelihood estimate of the mean number of microorganisms per unit volume may occur if censored data are ignored. It is also shown that the asymptotic variance of the maximum likelihood estimate may either be increased or decreased if censored data are ignored. Data from a trial of vaccines against pig pneumonia and data from an equine health certification process are used as illustrations.     

Recognition of Bisecting N-Acetylglucosamine: STRUCTURAL BASIS FOR ASYMMETRIC INTERACTION WITH THE MOUSE LECTIN DENDRITIC CELL INHIBITORY RECEPTOR 2*

Dendritic cell inhibitory receptor 2 (DCIR2) is a C-type lectin expressed on classical dendritic cells. We recently identified the unique ligand specificity of mouse DCIR2 (mDCIR2) toward biantennary complex-type glycans containing bisecting N-acetylglucosamine (GlcNAc). Here, we report the crystal structures of the mDCIR2 carbohydrate recognition domain in unliganded form as well as in complex with an agalactosylated complex-type N-glycan unit carrying a bisecting GlcNAc residue. Bisecting GlcNAc and the α1-3 branch of the biantennary oligosaccharide asymmetrically interact with canonical and non-canonical mDCIR2 residues. Ligand-protein interactions occur directly through mDCIR2-characteristic amino acid residues as well as via a calcium ion and water molecule. Our structural and biochemical data elucidate for the first time the unique binding mode of mDCIR2 for bisecting GlcNAc-containing glycans, a mode that contrasts sharply with that of other immune C-type lectin receptors such as DC-SIGN.     

金黄色葡萄球菌滤液制剂中肠毒素的检测研究

使用反向被动血凝试验(RPHA),对金黄色葡萄球菌滤液制剂中的肠毒素(SET)进行了检测研究.证明已生产和临床应用近10 a的金黄色葡萄球菌滤液制剂中检测到SET存在.其型别系属SET中的C型.按原生产工艺所生产的金黄色葡萄球菌滤液制剂原液中SET的含量在12.5～50 mg/L.生产时欲提高SET含量的条件试验中证实,使用葡萄球菌产毒的胰酪胨综合培养基,以摇瓶方式培养24～36 h,可使SET含量比用生产培养基培养提高16倍.金黄色萄萄球菌滤液制剂中SET的成分是极其稳定的,室温存放2 a未见该成分降低.其半衰期可达4 a.在采用RPHA和RPLA检测SET比较试验中证明:RPLA试剂的敏感性高于RPHA的4倍.但对于检测金黄色葡萄球菌滤液制剂中SET的实际含量几乎是相等的.     

Enhanced concanavalin a agglutination of trypsinised erythrocytes is due to a specific class of aggregation

Using the output of a rotational viscometer as a continuous index of aggregation, we have shown previously that the concanavalin A agglutination of native human erythrocytes can be resolved into three distinct classes of aggregation, static, type I and type II. Static aggregation occurs in the absence of shear forces while both type I and II aggregations are shear-induced. We now report that the increased concanavalin A agglutination of trypsinised erythrocytes is attributable to a specific enhancement in the development of type II aggregation. While type II formation in native cell suspensions requires high concanavalin A concentrations and continual shearing, an indistinguishable type of aggregation develops in suspensions of trypsinised red cells at considerably lower lectin concentrations and in the absence of applied shear forces.     

3-D structure modelling of the Staphylococcus simulans lipase: conformational changes, substrate specificity and novel structural features

We have modelled, using the CHARMM27 energy force field, the structures of closed and open forms of Staphylococcus simulans lipase (SSL) on the basis of the crystal structures of Bacillus stearothermophilus and Staphylococcus hyicus lipases, respectively. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. Superimposition of both closed and open forms of SSL allowed us to determine the hinge regions allowing the movements of the main and the second lid upon lipase activation. The flexibility of these hinge regions was checked by molecular dynamics simulations. The SSL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions.