α-Enolase, an Adhesion-Related Factor of Mycoplasma bovis

Mycoplasma bovis is the causative agent of Mycoplasma bovis-associated disease (MbAD). Although the mechanisms underlying M. bovis adherence to host cells is not clear, recent studies have shown that the cell surface protein α-enolase facilitates bacterial invasion and dissemination in the infected host. In this study, we cloned, expressed and purified recombinant M. bovis α-enolase and induced polyclonal anti-α-enolase antibodies in rabbits. M. bovis α-enolase was detected in the cytoplasmic and membrane protein fractions by these antibodies. Triple immunofluorescence labeling combined with confocal laser scanning microscopy (CLSM) revealed that the plasminogen (Plg) enhanced the adherence of M. bovis to embryonic bovine lung (EBL) cells; the values obtained for adherence and inhibition are consistent with this finding. Interestingly, we found that trace amounts of trypsin acted as a more effective enhancer of cell adherence than Plg. Hence, our data indicate that surface-associated M. bovis α-enolase is an adhesion-related factor of M. bovis that contributes to adherence by binding Plg

Assessment of drug entrapment within liposomes using photophysical probes

The photophysical and photochemical behavior of (R)-cinacalcet (CIN) and (S)-naproxen (NPX) entrapped within liposomes has been studied. For this purpose, liposome encapsulated drugs have been prepared through thin layer evaporation and characterized by transmission electron microscopy, cryoscopy scanning electron microscopy and dynamic light scattering. Steady state and time-resolved fluorescence experiments showed similar spectra, emission quantum yields, singlet energies and lifetimes for the selected drugs, outside and inside liposomes. By contrast, laser flash photolysis experiments revealed an important enhancement of the triplet lifetimes for entrapped drugs inside liposomes, indicating the spatial confinement existing in the microenvironment prevailing in these biomimetic entities. Thus, this photophysical property shows potential as a non-invasive, direct and valuable tool to monitor encapsulation of photoactive drugs and to probe the intraliposome environment. In addition, it provides a new quantitative indicator of the capability of liposomes to act as drug carriers.We gratefully acknowledge financial support from the Carlos III Institute of Health (Servet Contract CP11/00154 for I.A. and Red RETICS de investigacion de Reacciones Adversas a Alergenos y Farmacos, RIRAAF) and from Ministero dell'Istruzione, dell'Universita e della Ricerca (M.I.U.R. Italy).Oliverio, F.; Nuin Plá, NE.; Andreu Ros, MI.; Ragno, G.; Miranda Alonso, MÁ. (2014). Assessment of drug entrapment within liposomes using photophysical probes. European Journal of Pharmaceutics and Biopharmaceutics. 88(2):551-555. https://doi.org/10.1016/j.ejpb.2014.06.013S55155588

Invasion of Ureaplasma diversum in bovine spermatozoids

<p>Abstract</p> <p>Background</p> <p><it>Ureaplasma diversum </it>has been associated with infertility in cows. In bulls, this mollicute colonizes the prepuce and distal portion of the urethra and may infect sperm cells. The aim of this study is to analyze <it>in vitro </it>interaction of <it>U. diversum </it>isolates and ATCC strains with bovine spermatozoids. The interactions were observed by confocal microscopy and the gentamycin internalization assay.</p> <p>Findings</p> <p><it>U. diversum </it>were able to adhere to and invade spermatozoids after 30 min of infection. The gentamicin resistance assay confirmed the intracellularity and survival of <it>U. diversum </it>in bovine spermatozoids.</p> <p>Conclusions</p> <p>The intracellular nature of bovine ureaplasma identifies a new difficulty to control the reproductive of these animals.</p

The Interaction of Canine Plasminogen with Streptococcus pyogenes Enolase: They Bind to One Another but What Is the Nature of the Structures Involved?

For years it has been clear that plasminogen from different sources and enolase from different sources interact strongly. What is less clear is the nature of the structures required for them to interact. This work examines the interaction between canine plasminogen (dPgn) and Streptococcus pyogenes enolase (Str enolase) using analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), fluorescence polarization, dynamic light scattering (DLS), isothermal titration calorimetry (ITC), and simple pull-down reactions. Overall, our data indicate that a non-native structure of the octameric Str enolase (monomers or multimers) is an important determinant of its surface-mediated interaction with host plasminogen. Interestingly, a non-native structure of plasminogen is capable of interacting with native enolase. As far as we can tell, the native structures resist forming stable mixed complexes

N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60

Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1)-Pro(223)) and N2 (Gly(224)-Gly(411)), and the ADH region contains C1 (Gly(412)-Val(648)) and C2 (Pro(649)-Val(866)). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.The N2 subdomain exhibited the greatest affinity for Hsp60 with a K(D) of 9.50±2.6 nM. The K(D) of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies

Amyloids - A functional coat for microorganisms

Amyloids are filamentous protein structures ~10 nm wide and 0.1–10 µm long that share a structural motif, the cross-β structure. These fibrils are usually associated with degenerative diseases in mammals. However, recent research has shown that these proteins are also expressed on bacterial and fungal cell surfaces. Microbial amyloids are important in mediating mechanical invasion of abiotic and biotic substrates. In animal hosts, evidence indicates that these protein structures also contribute to colonization by activating host proteases that are involved in haemostasis, inflammation and remodelling of the extracellular matrix. Activation of proteases by amyloids is also implicated in modulating blood coagulation, resulting in potentially life-threatening complications.

Mycoplasma fermentans Binds to and Invades HeLa Cells: Involvement of Plasminogen and Urokinase

Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37°C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with β-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry

Low-visibility light-intensity laser-triggered release of entrapped calcein from 1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine liposomes is mediated through a type I photoactivation pathway

Amichai Yavlovich,1,* Mathias Viard,1,2,* Kshitij Gupta,1,* Jessica Sine,1,* Mylinh Vu,1 Robert Blumenthal,1 Darrell B Tata,3 Anu Puri1,*1Center for Cancer Research Nanobiology Program, National Cancer Institute, Frederick, MD, USA; 2Basic Science Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA; 3Centre for Devices and Radiological Health (CDRH)/Office of Science and Engineering Laboratories(OSEL)/Division of Physics, US Food and Drug Administration, White Oak, MD, USA*These authors contributed equally to this workAbstract: We recently reported on the physical characteristics of photo-triggerable liposomes containing dipalmitoylphosphatidylcholine (DPPC), and 1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) carrying a photo agent as their payload. When exposed to a low-intensity 514 nm wavelength (continuous-wave) laser light, these liposomes were observed to release entrapped calcein green (Cal-G; Ex/Em 490/517 nm) but not calcein blue (Cal-B; Ex/Em 360/460 nm). In this study, we have investigated the mechanism for the 514 nm laser-triggered release of the Cal-G payload using several scavengers that are known specifically to inhibit either type I or type II photoreaction pathways. Liposomes containing DPPC:DC8,9PC: distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)-2000 (86:10:04 mole ratio) were loaded either with fluorescent (calcein) or nonfluorescent (3H-inulin) aqueous markers. In addition, a non-photo-triggerable formulation (1-palmitoyl-2-oleoyl phosphatidylcholine [POPC]:DC8,9PC:DSPE-PEG2000) was also studied with the same payloads. The 514 nm wavelength laser exposure on photo-triggerable liposomes resulted in the release of Cal-G but not that of Cal-B or 3H-inulin, suggesting an involvement of a photoactivated state of Cal-G due to the 514 nm laser exposure. Upon 514 nm laser exposures, substantial hydrogen peroxide (H2O2, &asymp;100 &micro;M) levels were detected from only the Cal-G loaded photo-triggerable liposomes but not from Cal-B-loaded liposomes (&le;10 &micro;M H2O2). The Cal-G release from photo-triggerable liposomes was found to be significantly inhibited by ascorbic acid (AA), resulting in a 70%&ndash;80% reduction in Cal-G release. The extent of AA-mediated inhibition of Cal-G release from the liposomes also correlated with the consumption of AA. No AA consumption was detected in the 514 nm laser-exposed Cal B-loaded liposomes, thus confirming a role of photoactivation of Cal-G in liposome destabilization. Inclusion of 100 mM K3Fe(CN)6 (a blocker of electron transfer) in the liposomes substantially inhibited Cal-G release, whereas inclusion of 10 mM sodium azide (a blocker of singlet oxygen of type II photoreaction) in the liposomes failed to block 514 nm laser-triggered Cal-G release. Taken together, we conclude that low-intensity 514 nm laser-triggered release of Cal-G from photo-triggerable liposomes involves the type I photoreaction pathway.Keywords: visible laser-triggered payload release, photo-agents, photopolymerizable phospholipids, photodynamic actions, reactive oxygen specie

The surface-localised α-enolase of Mycoplasma suis is an adhesion protein

Mycoplasma suis belongs to the haemotrophic mycoplasmas which colonise red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. Due to the lack of a cultivation system, identification of adhesion structures has been difficult. So far, only one adhesion protein, i.e. MSG1 was identified. In order to determine further adhesion molecules of M. suis, we screened genomic M. suis libraries and performed Southern blot hybridisation analyses of genomic M. suis DNA. The α-enolase of M. suis was identified and analysed genetically and functionally. The encoding gene has 1623bp in size. The deduced amino acid sequence showed an overall identity of 59.6-65.1% to α-enolases of other pathogenic mycoplasmas. The 540aa M. suis α-enolase displays a size extension of about 90aa in comparison to α-enolases of other mycoplasmas. Recombinant α-enolase expressed in Escherichia coli demonstrated immunogenicity in experimentally infected pigs. Immunoblot, confocal laser scanning microscopy and immune electron microscopy analysis using antibodies against recombinant α-enolase, indicate the membrane and surface localisation of native α-enolase in M. suis, though no typical signal sequences exist. Furthermore, we showed that recombinant α-enolase binds to porcine erythrocyte lysate in a dose-dependent manner. E. coli transformants which express α-enolase on their surface acquire the ability to adhere to porcine red blood cells. In conclusion, our observations indicate that α-enolase could be involved in the adhesion of M. suis to porcine red blood cells