Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K _D of 108 ± 2 nM and has a high thermostability (T _m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.     

Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.     

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.     

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.     

桑叶水提浸膏的抑菌作用研究

研究桑叶水提浸膏对五种食品常见污染菌的抑菌作用。通过滤纸片扩散法测定其相对抑菌活性。结果表明,桑叶水提浸膏对金黄色葡萄球菌有较强的抑制作用,对大肠杆菌作用次之,对枯草芽孢杆菌、荧光假单孢杆菌、巨大芽孢杆菌抑菌效果较差。桑叶水提浸膏对金黄色葡萄球菌的最小抑菌浓度是5%,抑菌作用受pH和温度的影响较大,在酸性条件下抑菌效果较好,低于60℃处理样品对抑菌效果影响不大,但高于80℃处理,样品抑菌活性明显降低。     

Lead resistance and sensitivity in Staphylococcus aureus

Abstract Five lead-resistant strains of Staphylococcus aureus were isolated. Plasmid-free lead-sensitive variants were obtained from the three plasmid-bearing strains. Lead-resistant strains tolerated an approximately 600 × higher Pb(NO₃)₂ concentration than lead-sensitive strains. Both types of strains initially bound lead, but only the resistant strains accumulated the metal as an intracellular lead-phosphate.     

Notes on the almost unknown genus Jeotgalicoccus

Aims: To facilitate isolation and differentiation of the almost entirely unknown Jeotgalicoccus spp. Methods and Results: Jeotgalicoccus spp. have been found in dust samples using SSCP‐PCR analysis. As the cultivation of strains is necessary for further studies on virulence, pathogenicity or metabolism, we developed a method for cultural isolation and further differentiation of Jeotgalicoccus spp. We found that J. halotolerans, J. psychrophilus, J. marinus, as well as the related species Salinicoccus roseus grow on Baird Parker (BP) agar as black colonies without clear zones. J. pinnipedialis and S. jeotgali grow only weakly on BP agar without forming clearly delineated colonies. On BP agar, the colony‐forming Jeotgalicoccus and Salinicoccus spp. are not distinguishable from coagulase‐negative Staphylococcus spp. (CNS). However, unlike CNS, all of the above mentioned species are unreactive in the OF test. SSCP‐PCR was able to differentiate between all investigated Jeotgalicoccus and Salinicoccus spp., as all species had different band positions. Conclusions: Jeotgalicoccus spp. and Salinicoccus spp. may be widely distributed in the environment, but, until now, overlooked or confused with staphylococci. Further epidemiological studies, which are required to prove this hypothesis, are facilitated by the observations of our study. Significance and Impact: This not yet published information enables researchers to carry out epidemiological studies on Jeotgalicoccus spp. in a very cheap and easy way.