Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade I PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed. (C) 2008 Elsevier Masson SAS. All rights reserved.

Influence of surface texture on the galling characteristics of lean duplex and austenitic stainless steels

Two simulative test methods were used to study galling in sheet forming of two types of stainlesssteel sheet: austenitic (EN 1.4301) and lean duplex LDX 2101 (EN 1.4162) in different surface conditions. Thepin-on-disc test was used to analyse the galling resistance of different combinations of sheet materials and lubricants. The strip reduction test, a severe sheet forming tribology test was used to simulate the conditionsduring ironing. This investigation shows that the risk of galling is highly dependent on the surface texture of theduplex steel. Trials were also performed in an industrial tool used for high volume production of pumpcomponents, to compare forming of LDX 2101 and austenitic stainless steel with equal thickness. The forming forces, the geometry and the strains in the sheet material were compared for the same component.It was found that LDX steels can be formed to high strain levels in tools normally applied for forming ofaustenitic steels, but tool adaptations are needed to comply with the higher strength and springback of thematerial.

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.

Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy

We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy. The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins. We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores. We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM. The normal pore size found on the cell walls of American strains had diameters of about 200nm. Under conditions of oxidative stress the diameters changed to 400nm.This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress. This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry.

Ability of Salmonella spp. to Produce Biofilm Is Dependent on Temperature and Surface Material

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Infection of water buffalo in Rio de Janeiro Brazil with Anaplasma marginale strains also reported in cattle

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Incorporation of chlorhexidine gluconate or diacetate into a glass-ionomer cement: porosity, surface roughness, and anti-biofilm activity

To evaluate the porosity, surface roughness and anti-biofilm activity of a glass-ionomer cement (GIC) after incorporation of different concentrations of chlorhexidine (CHX) gluconate or diacetate. Methods: For the porosity and surface roughness tests, 10 test specimens were fabricated of the GIC Ketac Molar Easy Mix (KM) and divided into the following groups: Control, GIC and 0.5% CHX diacetate; GIC and 1.0% CHX diacetate; GIC and 2.0% CHX diacetate; GIC and 0.5% CHX gluconate; GIC and 1.0% CHX gluconate; GIC and 2.0% CHX gluconate. To evaluate porosity, the test specimens were fractured. The fragments were photographed by scanning electron microscopy (SEM), and the images analyzed with the aid of the software program Image J. The surface roughness (Ra) was obtained by the mean value of three readouts performed on the surface of each specimen, always through the center. To analyze the anti-biofilm activity, strains of S. mutans ATCC 35688 were used, and the groups control and GIC +CHX diacetate 1% were divided as follows: GIC (1 day); GIC (7 days), GIC (14 days), GIC (21 days); GIC+CHX (1 day), GIC+CHX (7 days), GIC+CHX (14 days), GIC+CHX (21 days); GIC+ CHX (1 day), GIC+ CHX (7 days), GIC+ CHX (14 days) and GIC+ CHX (21 days) using 10 test specimens per group. For biofilm growth, the specimens were placed in a vertical position in 24-well plates and incubated overnight 10 times. The culture medium was renewed every 24 hours. The suspension was diluted and seeded on BHI agar for quantification of the bacteria present. For evaluation of all the tests the two-way ANOVA was used, and if necessary, the Tukey test was applied, with a level of significance of 5%. Results: Regarding GIC porosity, the ANOVA showed that the presence of CHX increased the porosity (P< 0.001) proportionally to the increase in concentrations (P= 0.001), without however, presenting interaction between material and concentration (P= 0.705). Regarding the number of pores, a significant increase in pores was observed with the increase in CHX concentration (P= 0.003). The surface roughness test demonstrated no statistically significant effect as to increase or reduction in roughness at any of the CHX concentrations used (P> 0.05). Anti-biofilm activity analysis pointed out a significant effect of the factors material (P= 0.006) and time (P< 0.001), with CHX diacetate CHX presenting greater effectiveness in reducing microorganisms.

The Role of Dyslipidemia on Ocular Surface, Lacrimal and Meibomian Gland Structure and Function

Purpose: Dyslipidemia is characterized by high lipid blood levels that are risk factors for cardiovascular diseases, which are leading causes of death. However, it is unclear whether dyslipidemia is a cause of the dry eye syndrome (DES). Therefore we determined in transgenic mice models of dyslipidemia, whether there is an association with DES development. Methods: Dyslipidemic models included male and female adult mice overexpressing apolipoprotein CIII (Apo CIII), LDL receptor knockout (LDLR-KO) and ApoE knockout (ApoE-KO). They were compared with age-and gender-matched C57BL/6 mice. Ocular health was evaluated based on corneal slit lamp assessment, phenol red thread test (PRT) and impression cytology. Blood lipid profiles and histology of meibomian and lacrimal glands were also evaluated. Effects of high-fat diet and aging were observed in LDLR-KO and ApoCIII strains, respectively. Results: Body weight and lacrimal gland weight were significantly higher in male mice compared to females of the same strain (P < 0.05). Body weight was significantly lower in LDLRKO mice receiving high lipid diet compared to their controls (P = 0.0043). ApoE-KO were hypercholesterolemic and ApoCIII hypertriglyceridemic while LDLR-KO showed increases in both parameters. The PRT test was lower in male LDLR-KO mice with high-fat diet than control mice with standard diet (P = 0.0273). Aging did not affect lacrimal structural or functional parameters of ApoCIII strain. Conclusions: DES development is not solely dependent on dyslipidemia in relevant mice models promoting this condition. On the other hand, lacrimal gland structure and function are differentially impacted by lipid profile changes in male and female mice. This dissociation suggests that other factors beside dyslipidemia impact on tear film dysfunction and DES development.

Adsorption of Components of the Plasma Kinin-forming System on the Surface of Porphyromonas gingivalis Involves Gingipains as the Major Docking Platforms

Enhanced production of proinflammatory bradykinin-related peptides, the kinins, has been suggested to contribute to the pathogenesis of periodontitis, a common inflammatory disease of human gingival tissues. In this report, we describe a plausible mechanism of activation of the kinin-generating system, also known as the contact system or kininogen-kallikrein-kinin system, by the adsorption of its plasma-derived components such as high-molecular-mass kininogen (HK), prekallikrein (PK), and Hageman factor (FXII) to the cell surface of periodontal pathogen Porphyromonas gingivalis. The adsorption characteristics of mutant strains deficient in selected proteins of the cell envelope suggested that the surface-associated cysteine proteinases, gingipains, bearing hemagglutinin/adhesin domains (RgpA and Kgp) serve as the major platforms for HK and FXII adhesion. These interactions were confirmed by direct binding tests using microplate-immobilized gingipains and biotinylated contact factors. Other bacterial cell surface components such as fimbriae and lipopolysaccharide were also found to contribute to the binding of contact factors, particularly PK. Analysis of kinin release in plasma upon contact with P. gingivalis showed that the bacterial surface-dependent mechanism is complementary to the previously described kinin generation system dependent on HK and PK proteolytic activation by the gingipains. We also found that several P. gingivalis clinical isolates differed in the relative significance of these two mechanisms of kinin production. Taken together, these data show the importance of this specific type of bacterial surface-host homeostatic system interaction in periodontal infections.

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb O ) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.

Identification of an African Bacillus anthracis Lineage That Lacks Expression of the Spore Surface-Associated Anthrose-Containing Oligosaccharide

The surfaces of Bacillus anthracis endospores expose a pentasaccharide containing the monosaccharide anthrose, which has been considered for use as a vaccine or target for specific detection of the spores. In this study B. anthracis strains isolated from cattle carcasses in African countries where anthrax is endemic were tested for their cross-reactivity with monoclonal antibodies (MAbs) specific for anthrose-containing oligosaccharides. Unexpectedly, none of the isolates collected in Chad, Cameroon, and Mali were recognized by the MAbs. Sequencing of the four-gene operon encoding anthrose biosynthetic enzymes revealed the presence of premature stop codons in the aminotransferase and glycosyltransferase genes in all isolates from Chad, Cameroon, and Mali. Both immunological and genetic findings suggest that the West African isolates are unable to produce anthrose. The anthrose-deficient strains from West Africa belong to a particular genetic lineage. Immunization of cattle in Chad with a locally produced vaccine based on anthrose-positive spores of the B. anthracis strain Sterne elicited an anti-carbohydrate IgG response specific for a synthetic anthrose-containing tetrasaccharide as demonstrated by glycan microarray analysis. Competition immunoblots with synthetic pentasaccharide derivatives suggested an immunodominant role of the anthrose-containing carbohydrate in cattle. In West Africa anthrax is highly endemic. Massive vaccination of livestock in this area has taken place over long periods of time using spores of the anthrose-positive vaccine strain Sterne. The spread of anthrose-deficient strains in this region may represent an escape strategy of B. anthracis.

Emergence of Canine Distemper Virus Strains With Modified Molecular Signature and Enhanced Neuronal Tropism Leading to High Mortality in Wild Carnivores

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination-the classic presentation of CDV infection-was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.